Abstract:

A method of determining the presence, amount or concentration of at least
one autoantibody that reacts with neutrophil gelatinase-associated
lipocalin (NGAL), alone or in further combination with a method of
determining the concentration of NGAL, which methods can further comprise
diagnosing, prognosticating, or assessing the efficacy of a
therapeutic/prophylactic treatment of a patient and, optionally,
modifying the therapeutic/prophylactic treatment of the patient as needed
to improve efficacy; a kit comprising at least one component for assaying
a test sample for at least one autoantibody that reacts with NGAL and
instructions for assaying; a method of isolating an autoantibody that
reacts with NGAL; an isolated autoantibody that reacts with NGAL; and a
method for determining the reliability of an NGAL assay result.

Claims:

1. A method of determining the presence, amount or concentration of at
least one autoantibody that reacts with neutrophil gelatinase-associated
lipocalin (NGAL) or a fragment thereof in a test sample, which method
comprises assaying the test sample for at least one autoantibody that
reacts with NGAL (or a fragment thereof) by an assay employing NGAL (or a
fragment thereof) and at least one detectable label and comprising
comparing a signal generated by the detectable label as a direct or
indirect indication of the presence, amount or concentration of at least
one autoantibody that reacts with NGAL (or a fragment thereof) in the
test sample to a signal generated as a direct or indirect indication of
the presence, amount or concentration of an antibody that reacts with
NGAL (or a fragment thereof) in a control or calibrator, which is
optionally part of a series of calibrators in which each of the
calibrators differs from the other calibrators in the series by the
concentration of an antibody that reacts with NGAL (or a fragment
thereof), whereupon the presence, amount or concentration of at least one
autoantibody that reacts with NGAL (or a fragment thereof) in the test
sample is determined.

2. The method of claim 1, wherein the method comprises the following
steps:(i) contacting the test sample with NGAL (or a fragment thereof),
which comprises a detectable label and binds to at least one autoantibody
to form an NGAL (or a fragment thereof)/autoantibody complex, and(ii)
determining the presence, amount or concentration of at least one
autoantibody, which reacts with NGAL (or a fragment thereof), in the test
sample by detecting or measuring the signal generated by the detectable
label in the NGAL (or fragment thereof)/autoantibody complex formed in
(i), whereupon the presence, amount or concentration of at least one
autoantibody that reacts with NGAL (or a fragment thereof) in the test
sample is determined.

3. The method of claim 1, wherein the method comprises the following
steps:(i) contacting the test sample with NGAL (or a fragment thereof),
which binds to at least one autoantibody and which is optionally
immobilized on a solid phase, so as to form an NGAL (or a fragment
thereof)/autoantibody complex,(ii) contacting the NGAL (or a fragment
thereof)/autoantibody complex with at least one detection antibody, which
comprises a detectable label and binds to the autoantibody to form an
NGAL (or a fragment thereof)/autoantibody/detection antibody complex,
and(iii) determining the presence, amount or concentration of an
autoantibody, which reacts with NGAL (or fragment thereof), in the test
sample by detecting or measuring the signal generated by the detectable
label in the NGAL (or a fragment thereof)/autoantibody/detection antibody
complex formed in (ii), whereupon the presence, amount or concentration
of an autoantibody that reacts with NGAL (or fragment thereof) in the
test sample is determined,wherein the method optionally further comprises
removing any unbound at least one autoantibody after step (i) and
removing any unbound at least one detection antibody after step (ii).

4. The method of claim 1, wherein the detectable label is an acridinium
compound.

5. The method of claim 4, wherein the acridinium compound is an
acridinium-9-carboxamide or an acridinium-9-carboxylate aryl ester.

6. The method of claim 2, wherein the detectable label is an acridinium
compound.

7. The method of claim 6, wherein the acridinium compound is an
acridinium-9-carboxamide or an acridinium-9-carboxylate aryl ester.

8. The method of claim 3, wherein the detectable label is an acridinium
compound.

9. The method of claim 8, wherein the acridinium compound is an
acridinium-9-carboxamide or an acridinium-9-carboxylate aryl ester.

10. The method of claim 1, which further comprises previously,
simultaneously or subsequently determining the concentration of NGAL (or
a fragment thereof) in the test sample, which method comprises assaying
the test sample for NGAL (or a fragment thereof) by an assay employing at
least one specific binding partner for NGAL (or a fragment thereof) and
at least one detectable label and comprising comparing a signal generated
by the detectable label as a direct or indirect indication of the
concentration of NGAL (or a fragment thereof) in the test sample to a
signal generated as a direct or indirect indication of the concentration
of NGAL in a control or calibrator, which is optionally part of a series
of calibrators in which each of the calibrators differs from the other
calibrators in the series by the concentration of NGAL, whereupon the
concentration of NGAL (or a fragment thereof) in the test sample is
determined.

11. The method of claim 2, which further comprises previously,
simultaneously or subsequently determining the concentration of NGAL (or
a fragment thereof) in the test sample, which method comprises assaying
the test sample for NGAL (or a fragment thereof) by an assay employing at
least one specific binding partner for NGAL (or a fragment thereof) and
at least one detectable label and comprising comparing a signal generated
by the detectable label as a direct or indirect indication of the
concentration of NGAL (or a fragment thereof) in the test sample to a
signal generated as a direct or indirect indication of the concentration
of NGAL in a control or calibrator, which is optionally part of a series
of calibrators in which each of the calibrators differs from the other
calibrators in the series by the concentration of NGAL, whereupon the
concentration of NGAL (or a fragment thereof) in the test sample is
determined.

12. The method of claim 3, which further comprises previously,
simultaneously or subsequently determining the concentration of NGAL (or
a fragment thereof) in the test sample, which method comprises assaying
the test sample for NGAL (or a fragment thereof) by an assay employing at
least one specific binding partner for NGAL (or a fragment thereof) and
at least one detectable label and comprising comparing a signal generated
by the detectable label as a direct or indirect indication of the
concentration of NGAL (or a fragment thereof) in the test sample to a
signal generated as a direct or indirect indication of the concentration
of NGAL in a control or calibrator, which is optionally part of a series
of calibrators in which each of the calibrators differs from the other
calibrators in the series by the concentration of NGAL, whereupon the
concentration of NGAL (or a fragment thereof) in the test sample is
determined.

13. The method of claim 1, which further comprises diagnosing,
prognosticating, or assessing the efficacy of a therapeutic/prophylactic
treatment of a patient from whom the test sample was obtained, wherein,
if the method further comprises assessing the efficacy of a
therapeutic/prophylactic treatment of the patient from whom the test
sample was obtained, the method optionally further comprises modifying
the therapeutic/prophylactic treatment of the patient as needed to
improve efficacy.

14. The method of claim 2, which further comprises diagnosing,
prognosticating, or assessing the efficacy of a therapeutic/prophylactic
treatment of a patient from whom the test sample was obtained, wherein,
if the method further comprises assessing the efficacy of a
therapeutic/prophylactic treatment of the patient from whom the test
sample was obtained, the method optionally further comprises modifying
the therapeutic/prophylactic treatment of the patient as needed to
improve efficacy.

15. The method of claim 3, which further comprises diagnosing,
prognosticating, or assessing the efficacy of a therapeutic/prophylactic
treatment of a patient from whom the test sample was obtained, wherein,
if the method further comprises assessing the efficacy of a
therapeutic/prophylactic treatment of the patient from whom the test
sample was obtained, the method optionally further comprises modifying
the therapeutic/prophylactic treatment of the patient as needed to
improve efficacy.

16. The method of claim 10, which further comprises diagnosing,
prognosticating, or assessing the efficacy of a therapeutic/prophylactic
treatment of a patientfrom whom the test sample was obtained, wherein, if
the method further comprises assessing the efficacy of a
therapeutic/prophylactic treatment of the patient from whom the test
sample was obtained, the method optionally further comprises modifying
the therapeutic/prophylactic treatment of the patient as needed to
improve efficacy.

17. The method of claim 11, which further comprises diagnosing,
prognosticating, or assessing the efficacy of a therapeutic/prophylactic
treatment of a patient from whom the test sample was obtained, wherein,
if the method further comprises assessing the efficacy of a
therapeutic/prophylactic treatment of the patient from whom the test
sample was obtained, the method optionally further comprises modifying
the therapeutic/prophylactic treatment of the patient as needed to
improve efficacy.

18. The method of claim 12, which further comprises diagnosing,
prognosticating, or assessing the efficacy of a therapeutic/prophylactic
treatment of a patient from whom the test sample was obtained, wherein,
if the method further comprises assessing the efficacy of a
therapeutic/prophylactic treatment of the patient from whom the test
sample was obtained, the method optionally further comprises modifying
the therapeutic/prophylactic treatment of the patient as needed to
improve efficacy.

19. The method of claim 1, wherein the method is adapted for use in an
automated system or a semi-automated system.

20. The method of claim 2, wherein the method is adapted for use in an
automated system or a semi-automated system.

21. The method of claim 3, wherein the method is adapted for use in an
automated system or a semi-automated system.

22. The method of claim 10, wherein the method is adapted for use in an
automated system or a semi-automated system.

23. The method of claim 11, wherein the method is adapted for use in an
automated system or a semi-automated system.

24. The method of claim 12, wherein the method is adapted for use in an
automated system or a semi-automated system.

25. A kit for assaying a test sample for the presence, amount or
concentration of at least one autoantibody that reacts with NGAL (or a
fragment thereof) in a test sample, which kit comprises at least one
component for assaying the test sample for at least one autoantibody that
reacts with NGAL (or a fragment thereof) and instructions for assaying
the test sample for at least one autoantibody that reacts with NGAL (or a
fragment thereof), wherein the at least one component for assaying the
test sample for at least one autoantibody that reacts with NGAL (or a
fragment thereof) includes a composition comprising NGAL (or a fragment
thereof), which is optionally immobilized on a solid phase, and/or a
composition comprising an antibody that can bind to the at least one
autoantibody that reacts with NGAL (or a fragment thereof), wherein the
NGAL (or a fragment thereof) or the antibody is optionally detectably
labeled.

26. The kit of claim 25, which further comprises at least one component
for assaying the test sample for NGAL (or a fragment thereof) and
instructions for assaying the test sample for NGAL (or a fragment
thereof), wherein the at least one component for assaying the test sample
for NGAL (or a fragment thereof) includes a composition comprising a
specific binding partner for NGAL (or a fragment thereof), wherein the
specific binding partner for NGAL is optionally detectably labeled.

27. A method of isolating an autoantibody that reacts with NGAL, which
method comprises the steps of:(i) contacting NGAL (or a fragment thereof)
with a biological sample, which is known to contain an autoantibody that
reacts with NGAL (or a fragment thereof), wherein the NGAL (or a fragment
thereof) is optionally immobilized on a solid phase before or after
contact with the biological sample,(ii) isolating NGAL (or a fragment
thereof) to which is bound the autoantibody, and(iii) isolating the
autoantibody from the NGAL (or a fragment thereof),whereupon an
autoantibody that reacts with NGAL (or a fragment thereof) is isolated.

28. An isolated autoantibody that reacts with NGAL or a fragment thereof.

29. A method for determining the reliability of a result obtained from a
separate assay that was previously, simultaneously or subsequently
performed for detecting or quantifying the amount or concentration of
NGAL in a test sample obtained from a subject, wherein the method
comprises:(a) determining the amount or the concentration in the test
sample of at least one autoantibody that reacts with NGAL; and(b)
comparing the amount or the concentration in step (a) to a predetermined
level, wherein if the amount or the concentration is elevated as compared
to a predetermined level, then the amount or concentration of NGAL as
determined by separate assay is considered not to be reliable, and
wherein if the amount or the concentration in step (a) is lower or the
same as a predetermined level, then the amount or concentration of NGAL
as determined by separate assay is considered to be reliable.

Description:

TECHNICAL FIELD

[0001]The disclosure relates to isolated human autoantibodies and related
kits and methods for detecting a human autoantibody to NGAL or fragment
thereof, and determining the reliability of an NGAL assay result.

BACKGROUND

[0002]Neutrophil gelatinase-associated lipocalin (NGAL), which is also
known as human neutrophil lipocalin (HNL), N-formyl peptide binding
protein, and 25 kDa α2-microglobulin-related protein, is a 24 kDa
protein, which can exist as a monomer, a homodimer, or a heterodimer with
proteins, such as gelatinase B or matrix metalloproteinase-9 (MMP-9).
NGAL is a marker in the diagnosis and/or prognosis of a number of
diseases (see, e.g., Xu et al., Biochim. et Biophys. Acta 1482: 298-307
(2000)), disorders, and conditions, including inflammation, such as that
associated with infection. It is a marker for irritable bowel syndrome
(see, e.g., U.S. Pat. App. Pub. Nos. 2008/0166719 and 2008/0085524);
renal disorders, diseases and injuries (see, e.g., U.S. Pat. App. Pub.
Nos. 2008/0090304, 2008/0014644, 2008/0014604, 2007/0254370, and
2007/0037232); systemic inflammatory response syndrome (SIRS), sepsis,
severe sepsis, septic shock and multiple organ dysfunction syndrome
(MODS) (see, e.g., U.S. Pat. App. Pub. Nos. 2008/0050832 and
2007/0092911; see, also, U.S. Pat. No. 6,136,526); periodontal disease
(see, e.g., U.S. Pat. No. 5,866,432); and venous thromboembolic disease
(see, e.g., U.S. Pat. App. Pub. Nos. 2007/0269836), among others. In its
free, uncomplexed form it is a marker for ovarian cancer, invasive and
noninvasive breast cancer, and atypical ductal hyperplasia, which is a
major risk factor for breast cancer (see, e.g., U.S. Pat. App. Pub. No.
2007/0196876; see, also, U.S. Pat. Nos. 5,627,034 and 5,846,739 with
regard to assessing the proliferative status of a carcinoma). When
complexed with MMP-9, it also is a marker for conditions associated with
tissue remodeling (see, e.g., U.S. Pat. App. Pub. No. 2007/0105166 and
U.S. Pat. No. 7,153,660). A high level of NGAL (e.g., approximately 350
μg/L (Xu et al., Scand. J. Clin. Lab. Invest. 55: 125-131 (1995)) also
can be indicative of a bacterial infection as opposed to a viral
infection (see, e.g., U.S. Pat. App. Pub. No. 2004/0115728).

[0003]Levels of NGAL in the serum of a healthy human range from
approximately 50 μg/L to approximately 100 μg/L (Xu et al., J.
Immunol. Methods 171: 245-252 (1994); and Blaser et al., Clinica Chimica
Acta 235: 137-145 (1995)). Currently available assays for NGAL, such as
those used in diagnosis, prognosis, and assessment of therapeutic or
prophylactic efficacy, assess the level of NGAL in a test sample, such as
a fluid sample, in particular a sample of serum, plasma, or urine, from a
patient. Such assays compare the level of NGAL in the test sample to what
is considered to be normal, what is considered to be indicative of a
given condition, disorder, or disease state, or the level of NGAL in a
test sample taken from the patient previously. Based on such comparisons,
diagnoses, prognoses, and therapeutic/prophylactic treatment regimens
(e.g., active agent used or dosage of a given active agent) are
determined. Unfortunately, results obtained with such assays are subject
to interferences that may lead to aberrant results, for example,
false-negatives, false positives, or incorrect quantification, due to the
presence of heretofore unknown endogenous anti-NGAL antibodies.

[0004]In view of the foregoing, it is an object of the present disclosure
to provide an isolated human autoantibody to NGAL. It is another object
of the present disclosure to provide methods and kits for detecting
autoantibodies to NGAL in a test sample. Such methods and kits can be
used with methods and kits for the detection of NGAL and/or NGAL
complexed with another protein so as to minimize aberrant results due to
the presence of NGAL autoantibodies in the test sample and thereby
improve diagnoses, prognoses and assessments of therapeutic/prophylactic
efficacy. These and other objects and advantages, as well as additional
inventive features, will become apparent from the detailed description
provided herein.

SUMMARY

[0005]A method of determining the presence, amount or concentration of at
least one autoantibody that reacts with neutrophil gelatinase-associated
lipocalin (NGAL) or a fragment thereof in a test sample is provided. The
method comprises assaying the test sample for at least one autoantibody
that reacts with NGAL (or a fragment thereof) by an assay employing NGAL
(or a fragment thereof) and at least one detectable label and comprising
comparing a signal generated by the detectable label as a direct or
indirect indication of the presence, amount or concentration of at least
one autoantibody that reacts with NGAL (or a fragment thereof) in the
test sample to a signal generated as a direct or indirect indication of
the presence, amount or concentration of an antibody that reacts with
NGAL (or a fragment thereof) in a control or calibrator. The calibrator
is optionally part of a series of calibrators in which each of the
calibrators differs from the other calibrators in the series by the
concentration of an antibody that reacts with NGAL (or a fragment
thereof). The method can be adapted for use in an automated system or a
semi-automated system.

[0006]The method can comprise (i) contacting the test sample with NGAL (or
a fragment thereof), which comprises a detectable label and binds to at
least one autoantibody to form an NGAL (or fragment thereof)/autoantibody
complex, and (ii) determining the presence, amount or concentration of at
least one autoantibody, which reacts with NGAL (or a fragment thereof),
in the test sample by detecting or measuring the signal generated by the
detectable label in the NGAL (or fragment thereof)/autoantibody complex
formed in (i).

[0007]The method can comprise (i) contacting the test sample with NGAL (or
a fragment thereof), which binds to at least one autoantibody and which
is optionally immobilized on a solid phase, so as to form an NGAL (or a
fragment thereof)/autoantibody complex, (ii) contacting the NGAL (or a
fragment thereof)/autoantibody complex with at least one detection
antibody, which comprises a detectable label and binds to the
autoantibody to form an NGAL (or a fragment
thereof)/autoantibody/detection antibody complex, and (iii) determining
the presence, amount or concentration of an autoantibody, which reacts
with NGAL (or fragment thereof), in the test sample by detecting or
measuring the signal generated by the detectable label in the NGAL (or a
fragment thereof)/autoantibody/detection antibody complex formed in (ii).
The method optionally further comprises removing any unbound at least one
autoantibody after step (i) and removing any unbound at least one
detection antibody after step (ii).

[0008]The method can further comprise previously, simultaneously or
subsequently determining the concentration of NGAL (or a fragment
thereof) in the test sample. The method can comprise assaying the test
sample for NGAL (or a fragment thereof) by an assay employing at least
one specific binding partner for NGAL (or a fragment thereof) and at
least one detectable label and comprising comparing a signal generated by
the detectable label as a direct or indirect indication of the
concentration of NGAL (or a fragment thereof) in the test sample to a
signal generated as a direct or indirect indication of the concentration
of NGAL in a control or calibrator. The calibrator is optionally part of
a series of calibrators in which each of the calibrators differs from the
other calibrators in the series by the concentration of NGAL.

[0009]The method can further comprise diagnosing, prognosticating, or
assessing the efficacy of a therapeutic/prophylactic treatment of a
patient from whom the test sample was obtained. If the method further
comprises assessing the efficacy of a therapeutic/prophylactic treatment
of the patient from whom the test sample was obtained, the method
optionally further comprises modifying the therapeutic/prophylactic
treatment of the patient as needed to improve efficacy.

[0010]Also provided is a kit for assaying a test sample for the presence,
amount or concentration of at least one autoantibody that reacts with
NGAL (or a fragment thereof) in a test sample. The kit comprises at least
one component for assaying the test sample for at least one autoantibody
that reacts with NGAL (or a fragment thereof) and instructions for
assaying the test sample for at least one autoantibody that reacts with
NGAL (or a fragment thereof). The at least one component for assaying the
test sample for at least one autoantibody that reacts with NGAL (or a
fragment thereof) includes a composition comprising NGAL (or a fragment
thereof), which is optionally immobilized on a solid phase, and/or a
composition comprising an antibody that can bind to the at least one
autoantibody that reacts with NGAL (or a fragment thereof), wherein the
NGAL (or a fragment thereof) or the antibody is optionally detectably
labeled.

[0011]Further provided is a method of isolating an autoantibody that
reacts with NGAL. The method comprises (i) contacting NGAL (or a fragment
thereof) with a biological sample, which is known to contain an
autoantibody that reacts with NGAL, wherein the NGAL is optionally
immobilized on a solid phase before or after contact with the biological
sample, (ii) isolating NGAL to which is bound the autoantibody, and (iii)
isolating the autoantibody from the NGAL. Still further provided is an
isolated autoantibody that reacts with NGAL.

DETAILED DESCRIPTION

[0012]The present disclosure is based, at least in part, on the surprising
and unexpected discovery of endogenous antibodies, i.e., autoantibodies,
in human serum and plasma that react with neutrophil
gelatinase-associated lipocalin (NGAL).

Definitions

[0013](a) "Neutrophil gelatinase-associated lipocalin (NGAL)," which is
also known as human neutrophil lipocalin (HNL), N-formyl peptide binding
protein, and 25 kDa α2-microglobulin-related protein, is a 24 kDa
protein, which can exist as a monomer, a homodimer, or a heterodimer with
proteins, such as gelatinase B or matrix metalloproteinase-9 (MMP-9).
See, e.g., Kjeldsen et al., J. Biol. Chem. 268 (14): 10425-10432 (1993),
for an exemplary amino acid sequence. Generally, when present, the signal
peptide comprises amino acids 1-20. Numbering as applied herein is
considering a signal peptide at residues 1-20, with the understanding
that such a signal peptide may or may not be present.

[0014]The NGAL polynucleotide or polypeptide can be any NGAL sequence,
e.g., including that set forth as Genbank accession numbers Genpept
CAA58127 (SEQ ID NO:1), AAB26529, XP--862322, XP--548441,
P80108, P11672, X83006.1, X99133.1, CAA67574.1, BC033089.1, AAH33089.1,
S75256.1, AD14168.1, JC2339, 1DFVA, 1DFVB, 1L6MA, 1L6MB, 1L6MC, 1NGLA,
1QQSA, 1X71A, 1X71B, 1X71C, 1X89A, 1X89B, 1X89C, 1X8UA, 1X8UB, and 1X8UC.
NGAL polynucleotide and polypeptide (e.g., polyamino acid) sequences are
as found in nature, based on sequences found in nature, isolated,
synthetic, semi-synthetic, recombinant, or other. In one embodiment, the
NGAL is human NGAL (also known as "hNGAL"). NGAL polypeptide sequences
can be of the mature human NGAL sequence (sequence not including the
20-residue amino acid signal peptide typically found in nature, and/or
minus any other signal peptide sequence). When a signal peptide is
present, it is numbered, e.g., as residues 1 to 20, with comparable
numbering applied for the encoding polynucleotide sequence.

[0015]Likewise, an initial Met residue at the N-terminus of NGAL is
present only in NGAL produced in prokaryotes (e.g., E. coli), or in
synthetic (including semi-synthetic) or derived sequences, and not in
NGAL produced in eukaryotes (e.g., mammalian cells, including human and
yeast cells). Consequently, when present, an initial Met residue is
typically counted as a negative number, e.g., as residue -1, with no
similar numbering adjustment being made for the polynucleotide sequence
in a prokaryotic versus eukaryotic background or expression system
inasmuch as the polynucleotide sequence is replicated and transcribed the
same in both backgrounds and the difference lies at the level of
translation.

[0016]Accordingly, the disclosure herein encompasses a multitude of
different NGAL polynucleotide and polypeptide sequences as present and/or
produced in a prokaryotic and/or eukaryotic background (e.g., with
consequent optimization for codon recognition). In sum, the sequences may
or may not possess or encode: (a) a signal peptide; (b) an initiator Met
residue present in the mature NGAL sequence at the N-terminus; (c) an
initiator Met residue present at the start of a signal peptide that
precedes the mature NGAL protein; and (d) other variations such as would
be apparent to one skilled in the art.

[0017]Exemplary sequences include, but are not limited to, those as set
forth herein: SEQ ID NO: 1 (NGAL wild-type polypeptide including signal
peptide); SEQ ID NO:2 (NGAL wild-type polypeptide not including any
signal peptide, and which can be preceded by a Met initiator residue when
produced in prokaryotes and a Met initiator codon is present; however,
there is no Met initiator residue when produced in eukaryotes, regardless
of whether a Met initiator codon is present); and SEQ ID NO:3 (NGAL
wild-type polynucleotide sequence including that encoding a signal
peptide). Exemplary sequences further include any mutant sequences set
forth in any one or more of U.S. Provisional Application Nos. 60/981,470,
60/981,471 and 60/981,473, all filed on Oct. 19, 2007, and U.S. patent
application Ser. Nos. 12/104,408, 12/104,410, and 12/104,413, all filed
on Apr. 16, 2008, and each of which are incorporated by reference in
their entireties for their teachings regarding same.

[0021](b) "NGAL analog" refers to a biologically active analog of NGAL,
such as a biologically active analog of human NGAL. The analog can
comprise truncations, deletions, insertions, substitutions, replacements,
side-chain extensions, and fusions, such as a fusion with another
protein, as well as combinations of any of the foregoing, which do not
eliminate the biological activity of NGAL. Exemplary NGAL analogs include
but are not limited to any mutated NGAL sequences set forth in any one or
more of U.S. Provisional Application Nos. 60/981,470, 60/981,471 and
60/981,473, all filed on Oct. 19, 2007, and U.S. patent application Ser.
Nos. 12/104,408, 12/104,410, and 12/104,413, all filed on Apr. 16, 2008,
and each of which are incorporated by reference in their entireties for
their teachings regarding same.

[0022](c) "NGAL conjugate" refers to NGAL or a fragment thereof that
includes at least one modifying moiety or at least one reactive entity
attached thereto. Examples of modifying moieties include, but are not
limited to, moieties that affect stability, solubility, and/or biological
activity (e.g., hydrophilic polymers or oligomers, amphiphilic polymers
or oligomers, and lipophilic polymers or oligomers), hydrophilic
moieties, polyethylene glycol moieties, biocompatible water-soluble
moieties, polycationic moieties, amphiphilic moieties, polyethylene
glycol/alkyl-modifying moieties, etc. Such modifying moieties can be
covalently bonded to NGAL or a fragment thereof. The modifying moiety can
form a covalent bond with a component of blood, such as a blood protein,
e.g., by way of an amino group, a hydroxyl group, or a thiol group
present on the component. The amino group can form a covalent bond with
reactive entities like carboxy, phosphoryl or acyl; the hydroxyl group
can form a covalent bond with reactive entities like activated esters;
and the thiol group can form a covalent bond with reactive entities like
esters or mixed anhydrides. The preferred blood components are mobile
blood components like serum albumin, immunoglobulins, or combinations
thereof, and the preferred reactive entity comprises an anhydride like
maleimide or a maleimide-containing group.

[0023](d) "NGAL derivative" refers to an NGAL analog, an NGAL conjugate,
or a recombinant form of NGAL.

[0024](e) "Control" refers to a composition known to not contain an
antibody to NGAL ("negative control") or to contain an antibody to NGAL
("positive control"). A positive control can comprise a known
concentration of an antibody to NGAL. "Control" and "positive control"
may be used interchangeably herein to refer to a composition comprising a
known concentration of an antibody to NGAL. A "positive control" can be
used to establish assay performance characteristics and is a useful
indicator of the integrity of reagents (e.g., analytes).

[0025](f) "Series of calibrating compositions" refers to a plurality of
compositions comprising a known concentration of an antibody to NGAL,
wherein each of the compositions differs from the other compositions in
the series by the concentration of the antibody to NGAL.

[0026](g) In the context of immunoassays and kits described herein,
"quality control reagents" include, but are not limited to, calibrators,
controls, and sensitivity panels. A "calibrator" or "standard" typically
is used (e.g., one or more, such as a plurality) in order to establish
calibration (standard) curves for interpolation of the concentration of
an analyte, such as an antibody or an analyte. Alternatively, a single
calibrator, which is near a predetermined positive/negative cutoff, can
be used. Multiple calibrators (i.e., more than one calibrator or a
varying amount of calibrator(s)) can be used in conjunction so as to
comprise a "sensitivity panel."

[0027](h) "Predetermined cutoff" and "predetermined level" refer generally
to an assay cutoff value that is used to assess
diagnostic/prognostic/therapeutic efficacy results by comparing the assay
results against the predetermined cutoff/level, where the predetermined
cutoff/level already has been linked or associated with various clinical
parameters (e.g., severity of disease,
progression/nonprogression/improvement, etc.). The present disclosure
provides exemplary predetermined levels. However, it is well-known that
cutoff values may vary depending on the nature of the immunoassay (e.g.,
antibodies employed, etc.). It further is well within the ordinary skill
of one in the art to adapt the disclosure herein for other immunoassays
to obtain immunoassay-specific cutoff values for those other immunoassays
based on this disclosure. Whereas the precise value of the predetermined
cutoff/level may vary between assays, the correlations as described
herein should be generally applicable.

[0028](i) "Sample," "test sample," and "patient sample" may be used
interchangeably herein. The sample, such as a sample of urine, serum, or
plasma, can be obtained from a subject or patient using routine
techniques known in the art. The sample can be used directly as obtained
from a patient or can be pre-treated, such as by filtration,
distillation, extraction, concentration, centrifugation, inactivation of
interfering components, addition of reagents, and the like, to modify the
character of the sample in some manner as discussed herein or otherwise
as is known in the art.

[0029](j) "Patient" and "subject" may be used interchangeably herein to
refer to an animal, such as a bird (e.g., a duck or a goose), a shark, a
whale, and a mammal, including a non-primate (for example, a cow, pig,
camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog,
rat, and mouse) and a primate (for example, a monkey, a chimpanzee, and a
human). Preferably, the patient or subject is a human, such as a human at
risk for cardiovascular disease, a human having cardiovascular disease, a
human at risk for renal disease, or a human having renal disease.

[0030](k) "At least one component," "component," and "components" refer
generally to a capture antibody, a detection or conjugate antibody, a
calibrator, a control, a sensitivity panel, a container, a buffer, a
diluent, a salt, an enzyme, a co-factor for an enzyme, a detection
reagent, a pretreatment reagent/solution, a substrate (e.g., as a
solution), a stop solution, and the like that can be included in a kit
for assay of a test sample, such as a patient urine, serum or plasma
sample, in accordance with the methods described herein and other methods
known in the art. Some components can be in solution or lyophilized for
reconstitution for use in an assay.

[0031](l) "Antibody" (Ab) and "antibodies" (Abs) refer to monoclonal
antibodies, multispecific antibodies, human antibodies, humanized
antibodies (fully or partially humanized), animal antibodies (such as,
but not limited to, a bird (for example, a duck or a goose), a shark, a
whale, and a mammal, including a non-primate (for example, a cow, pig,
camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog,
rat, mouse, etc.) or a non-human primate (for example, a monkey, a
chimpanzee, etc.), recombinant antibodies, chimeric antibodies,
single-chain Fvs ("scFv"), single chain antibodies, single domain
antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments,
disulfide-linked Fvs ("sdFv"), and anti-idiotypic ("anti-Id") antibodies,
dual-domain antibodies (e.g., dual variable domain antibodies, or
DVD-IgGs), and functionally active epitope-binding fragments of any of
the above. In particular, antibodies include immunoglobulin molecules and
immunologically active fragments of immunoglobulin molecules, namely,
molecules that contain an analyte-binding site. Immunoglobulin molecules
can be of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), class
(for example, IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2), or subclass. An antibody, whose affinity (namely, KD, kd or
ka) has been increased or improved via the screening of a combinatory
antibody library that has been prepared using bio-display, is referred to
as an "affinity maturated antibody." For simplicity sake, an antibody
against an analyte is frequently referred to herein as being either an
"anti-analyte antibody" or an "analyte antibody" (e.g., an anti-NGAL
antibody or an NGAL antibody).

[0032](m) "Autoantibody" and "autoantibodies" refer to endogenous
antibodies that bind to (or "react with") an analyte that occurs
naturally in the animal in which the antibody is produced. In the context
of the present disclosure, the analyte is NGAL or a fragment thereof. Use
of "at least one" and "one or more" with respect to autoantibodies means
at least one or one or more types or populations of autoantibodies, i.e.,
such as autoantibodies that react with different epitopes on NGAL or
fragments thereof. For simplicity sake, an autoantibody against an
analyte is frequently referred to herein as being either an "anti-analyte
autoantibody" or an "analyte autoantibody" (e.g., an anti-NGAL
autoantibody or an NGAL autoantibody).

[0033](n) "Label" and "detectable label" mean a moiety attached to an
antibody or an analyte to render the reaction between the antibody and
the analyte detectable, and the antibody or analyte so labeled is
referred to as "detectably labeled." A label can produce a signal that is
detectable by visual or instrumental means. Various labels include
signal-producing substances, such as chromogens, fluorescent compounds,
chemiluminescent compounds, radioactive compounds, and the like.
Representative examples of labels include moieties that produce light,
e.g., acridinium compounds, and moieties that produce fluorescence, e.g.,
fluorescein. Other labels are described herein.

[0036](q) "Tracer" means an analyte or analyte fragment conjugated to a
label, such as NGAL (or a fragment thereof) conjugated to a label, e.g.,
a fluorescein moiety, wherein the analyte conjugated to the label can
effectively compete with the analyte for sites on an antibody specific
for the analyte.

[0037](r) A "solid phase" refers to any material that is insoluble, or can
be made insoluble by a subsequent reaction. The solid phase can be chosen
for its intrinsic ability to attract and immobilize a capture agent.
Alternatively, the solid phase can have affixed thereto a linking agent
that has the ability to attract and immobilize the capture agent. The
linking agent can, for example, include a charged substance that is
oppositely charged with respect to the capture agent itself or to a
charged substance conjugated to the capture agent. In general, the
linking agent can be any binding partner (preferably specific) that is
immobilized on (attached to) the solid phase and that has the ability to
immobilize the capture agent through a binding reaction. The linking
agent enables the indirect binding of the capture agent to a solid phase
material before the performance of the assay or during the performance of
the assay. The solid phase can, for example, be plastic, derivatized
plastic, magnetic or non-magnetic metal, glass or silicon, including, for
example, a test tube, microtiter well, sheet, bead, microparticle, chip,
and other configurations known to those of ordinary skill in the art.

[0038](s) "Specific binding partner" is a member of a specific binding
pair. A specific binding pair comprises two different molecules, which
specifically bind to each other through chemical or physical means.
Therefore, in addition to antigen and antibody specific binding pairs of
common immunoassays, other specific binding pairs can include biotin and
avidin (or streptavidin), carbohydrates and lectins, complementary
nucleotide sequences, effector and receptor molecules, cofactors and
enzymes, enzyme inhibitors, and enzymes and the like. Furthermore,
specific binding pairs can include members that are analogs of the
original specific binding members, for example, an analyte-analog.
Immunoreactive specific binding members include antigens, antigen
fragments, and antibodies, including monoclonal and polyclonal antibodies
as well as complexes and fragments thereof, whether isolated or
recombinantly produced.

[0040](u) "Cardiovascular disease" refers to various clinical diseases,
disorders or conditions involving the heart, blood vessels or
circulation. The diseases, disorders or conditions can be due to
atherosclerotic impairment of coronary, cerebral or peripheral arteries.
Cardiovascular disease includes, but is not limited to, coronary artery
disease, peripheral vascular disease, atherosclerosis, hypertension,
myocardial infarction (i.e., heart attack, e.g., primary or secondary,
which occurs when an area of heart muscle dies or is damaged because of
an inadequate supply of oxygen to that area), myocarditis, acute coronary
syndrome, angina pectoris (i.e., chest discomfort caused by inadequate
blood flow through the blood vessels (coronary vessels) of the
myocardium), sudden cardiac death, cerebral infarction, restenosis,
syncope, ischemia, transient ischemic attack, reperfusion injury,
vascular occlusion, carotid obstructive disease, cardiovascular
autoimmune disease, etc. By "cardiovascular autoimmune disease" is meant
any deviation from a healthy or normal condition of the heart that is due
to an underlying autoimmune disease, including any structural or
functional abnormality of the heart, or of the blood vessels supplying
the heart, that impairs typical functioning. Examples of cardiovascular
autoimmune diseases include myocarditis, cardiomyopathy, and ischemic
heart disease, each due to an underlying autoimmune disease.
"Myocarditis" refers to inflammation of the myocardium. Myocarditis can
be caused by a variety of conditions, such as viral infection,
sarcoidosis, rheumatic fever, autoimmune diseases (such as systemic lupus
erythematosus, etc.), and pregnancy. "Cardiomyopathy" refers to a
weakening of the heart muscle or a change in heart muscle structure. It
is often associated with inadequate heart pumping or other heart function
abnormalities. Cardiomyopathy can be caused by viral infections, heart
attacks, alcoholism, long-term, severe high blood pressure, nutritional
deficiencies (particularly selenium, thiamine, and L-camitine), systemic
lupus erythematosus, celiac disease, and end-stage kidney disease. Types
of cardiomyopathy include dilated cardiomyopathy, hypertrophic
cardiomyopathy, and restrictive cardiomyopathy. "Dilated cardiomyopathy"
refers to a global, usually idiopathic, myocardial disorder characterized
by a marked enlargement and inadequate function of the left ventricle.
Dilated cardiomyopathy includes ischemic cardiomyopathy, idiopathic
cardiomyopathy, hypertensive cardiomyopathy, infectious cardiomyopathy,
alcoholic cardiomyopathy, toxic cardiomyopathy, and peripartum
cardiomyopathy. "Hypertrophic cardiomyopathy" refers to a condition
resulting from the right and left heart muscles growing to be different
sizes. "Restrictive cardiomyopathy" refers to a condition characterized
by the heart muscle's inability to relax between contractions, which
prevents it from filling sufficiently. "Ischemic heart disease" refers to
any condition in which heart muscle is damaged or works inefficiently
because of an absence or relative deficiency of its blood supply; most
often caused by atherosclerosis, it includes angina pectoris, acute
myocardial infarction, and chronic ischemic heart disease.

[0041](v) "Heart failure" refers to a condition in which the heart cannot
pump blood efficiently to the rest of the body. Heart failure can be due
to damage to the heart or narrowing of the arteries due to infarction,
cardiomyopathy (primary or secondary), hypertension, coronary artery
disease, valve disease, birth defects or infection. Heart failure can
further be described as chronic, congestive, acute, decompensated,
systolic or diastolic. The New York Heart Association (NYHA)
classification describes the severity of the disease based on functional
capacity of the patient; NYHA class can progress and/or regress based on
treatment or lack of response to treatment. In heart failure, "increased
severity" of cardiovascular disease refers to the worsening of disease as
indicated by increased NYHA classification, to, for example, Class III or
Class IV, and "reduced severity" of cardiovascular disease refers to an
improvement of the disease as indicated by reduced NYHA classification,
from, for example, class III or IV to class II or I.

[0042](w) "Autoimmune disease" refers to the loss of immunological
tolerance to self antigens. Some criteria for a diagnosis of autoimmune
disease include: (1) the presence of circulating autoantibodies; (2)
autoantibodies observed in the affected organ; (3) target antigen
identified; (4) inducible in an animal model either by immunization with
antigen, serum, or autoantibody transfer; and (5) responsive to
immunosuppressive therapy or immunoabsorption. Other characteristics of
autoimmune disease include its: (a) increased prevalence in women; (b)
familial clustering (although this varies with disease); (c) asymptomatic
risk (i.e., the presence of autoantibodies may precede the disease by
years); (d) periodic nature; and (e) chronic nature.

[0043](x) "Autoimmunity" refers to one or more immune responses directed
against host antigens, characterized, for example, by the presence of
autoantibodies or T lymphocytes reactive with host antigens.

[0045](z) "Risk" refers to the possibility or probability of a particular
event occurring either presently, or, at some point in the future. "Risk
stratification" refers to an array of known clinical risk factors that
allows physicians to classify patients into a low, moderate, high or
highest risk of developing a particular disease, disorder or condition.

[0046](aa) "About" refers to approximately a +/-10% variation from the
stated value. It is to be understood that such a variation is always
included in any given value provided herein, whether or not specific
reference is made to it.

[0047](bb) As used herein, the term "reliability" means that with respect
to a given result or value (such as that obtained from an assay, such as
an immunoassay or a chemiluminescent assay) that there is at least about
a 90% certainty (e.g., from about a 90% certainty to about a 100%
certainty) that said result or value is accurate or correct, preferably
at least about a 95% certainty (e.g., from about a 95% certainty to about
a 100% certainty) that said result or value is accurate or correct.

[0048]The above terminology is provided for the purpose of describing
particular embodiments. The terminology is not intended to be limiting.

Method for Determining the Presence, amount or concentration of at Least
One Autoantibody that Reacts with NGAL (or a Fragment Thereof) in a Test
Sample

[0049]The present disclosure provides a method for determining the
presence, amount or concentration of at least one autoantibody that
reacts with NGAL (or a fragment thereof) in a test sample. As indicated
previously herein, the presence of autoantibodies to NGAL (or fragments
thereof) in a test sample can contribute to the generation of false
negative results obtained in an assay. The method of the present
disclosure enables one to determine the presence, amount or concentration
of one or more autoantibodies in a test sample before, at the same time
as, or after performing an assay for NGAL. The accuracy or reliability of
results previously obtained with an assay for NGAL can be assessed with a
method of determining the presence, amount or concentration of at least
one autoantibody that reacts with NGAL (or a fragment thereof) in a test
sample in accordance with the present disclosure.

[0050]The method can be performed in a homogeneous or heterogeneous
format. It will be recognized by those skilled in the art that an
essential difference between the two formats exists. For example,
homogeneous formats lack one or more steps to separate a complex between
an analyte of interest in a test sample and a specific binding partner
for the analyte of interest from uncomplexed binding partners and other
components of a test sample. Further, homogeneous assays employ
detectable labels. One or more characteristics of the signal generated
from the detectable label is/are modulated by the formation of a complex
between the analyte of interest in the test sample (i.e., an autoantibody
to NGAL (or a fragment thereof)) and a specific binding partner for the
analyte of interest (e.g., NGAL (or a fragment thereof)). Examples of
such homogeneous assays that can be used include, but are not limited to,
fluorescence polarization immunoassay (FPIA), enzyme multiplied
immunoassay technique (EMIT), bioluminescence resonance energy transfer
(BRET), homogeneous chemiluminescent assay, etc. In a homogeneous format,
after the test sample is obtained from a subject, a first mixture is
prepared. The mixture contains the test sample being assessed for
autoantibodies to NGAL (or a fragment thereof) and a first specific
binding partner that is labeled with a detectable label. The first
specific binding partner can be NGAL (or a fragment thereof).

[0051]Any suitable detectable label as is known in the art can be used.
For example, a fluorescent label can be used in FPIA (see, e.g., U.S.
Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5352803, which
are hereby incorporated by reference in their entireties). An acridinium
compound can be used as a detectable label in a homogeneous
chemiluminescent assay (see, e.g., Adamczyk et al., Bioorg. Med. Chem.
Lett. 16: 1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett. 4:
2313-2317 (2004); Adamczyk et al., Biorg. Med. Chem. Lett. 14: 3917-3921
(2004); and Adamczyk et al., Org. Lett. 5: 3779-3782 (2003)). Preferably,
the acridinium compound is an acridinium-9-carboxamide. Specifically, the
acridinium-9-carboxamide has a structure according to formula I:

[0053]An example of an acridinium-9-carboxylate aryl ester of formula II
is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available
from Cayman Chemical, Ann Arbor, Mich.). Methods for preparing acridinium
9-carboxylate aryl esters are described in McCapra et al., Photochem.
Photobiol. 4: 1111-21 (1965); Razavi et al., Luminescence 15: 245-249
(2000); Razavi et al., Luminescence 15: 239-244 (2000); and U.S. Pat. No.
5,241,070 (each incorporated herein by reference in their entireties for
their teachings regarding same). Such acridinium-9-carboxylate aryl
esters are efficient chemiluminescent indicators for hydrogen peroxide
produced in the oxidation of an analyte by at least one oxidase in terms
of the intensity of the signal and/or the rapidity of the signal. The
course of the chemiluminescent emission for the acridinium-9-carboxylate
aryl ester is completed rapidly, i.e., in under 1 second, while the
acridinium-9-carboxamide chemiluminescent emission extends over 2
seconds. Acridinium-9-carboxylate aryl ester, however, loses its
chemiluminescent properties in the presence of protein. Therefore, its
use requires the absence of protein during signal generation and
detection. Methods for separating or removing proteins in the sample are
well-known to those skilled in the art and include, but are not limited
to, ultrafiltration, extraction, precipitation, dialysis, chromatography,
and/or digestion (see, e.g., Wells, High Throughput Bioanalytical Sample
Preparation. Methods and Automation Strategies, Elsevier (2003)). The
amount of protein removed or separated from the test sample can be about
40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,
about 75%, about 80%, about 85%, about 90%, or about 95%. Further details
regarding acridinium-9-carboxylate aryl ester and its use are set forth
in U.S. patent application Ser. No. 11/697,835, filed Apr. 9, 2007.
Acridinium-9-carboxylate aryl esters can be dissolved in any suitable
solvent, such as degassed anhydrous N,N-dimethylformamide (DMF) or
aqueous sodium cholate.

[0055]"Alkenyl" means a straight or branched chain hydrocarbon containing
from 2 to 10 carbons and containing at least one carbon-carbon double
bond formed by the removal of two hydrogens. Representative examples of
alkenyl include, but are not limited to, ethenyl, 2-propenyl,
2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl,
2-methyl-1-heptenyl, and 3-decenyl.

[0056]"Alkynyl" means a straight or branched chain hydrocarbon group
containing from 2 to 10 carbon atoms and containing at least one
carbon-carbon triple bond. Representative examples of alkynyl include,
but are not limited, to acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl,
2-pentynyl, and 1-butynyl.

[0057]"Aryalkyl" means an aryl group, as defined herein, appended to the
parent molecular moiety through an alkyl group, as defined herein.
Representative examples of arylalkyl include, but are not limited to,
benzyl, 2-phenylethyl, 3-phenylpropyl, and 2-naphth-2-ylethyl.

[0058]"Aryl" means a phenyl group, or a bicyclic or tricyclic fused ring
system in which one or more of the fused rings is a phenyl group.
Bicyclic fused ring systems are exemplified by a phenyl group fused to a
cycloalkenyl group, as defined herein, a cycloalkyl group, as defined
herein, or another phenyl group. Tricyclic fused ring systems are
exemplified by a bicyclic fused ring system fused to a cycloalkenyl
group, as defined herein, a cycloalkyl group, as defined herein, or
another phenyl group. Representative examples of aryl include, but are
not limited to, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl,
naphthyl, phenyl, and tetrahydronaphthyl. The aryl groups of the present
invention can be optionally substituted with one, two, three, four, or
five substituents independently selected from the group consisting of
alkoxy, alkyl, carboxyl, halo, and hydroxyl.

[0059]"Cycloalkenyl" refers to a non-aromatic cyclic or bicyclic ring
system having from three to ten carbon atoms and one to three rings,
wherein each five-membered ring has one double bond, each six-membered
ring has one or two double bonds, each seven- and eight-membered ring has
one to three double bonds, and each nine- to ten-membered ring has one to
four double bonds. Representative examples of cycloalkenyl groups include
cyclohexenyl, octahydronaphthalenyl, norbornylenyl, and the like. The
cycloalkenyl groups can be optionally substituted with one, two, three,
four, or five substituents independently selected from the group
consisting of alkoxy, alkyl, carboxyl, halo, and hydroxyl.

[0060]"Cycloalkyl" refers to a saturated monocyclic, bicyclic, or
tricyclic hydrocarbon ring system having three to twelve carbon atoms.
Representative examples of cycloalkyl groups include cyclopropyl,
cyclopentyl, bicyclo[3.1.1]heptyl, adamantyl, and the like. The
cycloalkyl groups of the present invention can be optionally substituted
with one, two, three, four, or five substituents independently selected
from the group consisting of alkoxy, alkyl, carboxyl, halo, and hydroxyl.

[0061]"Sulfoalkyl" refers to an alkyl group to which a sulfonate group is
bonded, wherein the alkyl is bonded to the molecule of interest, whereas
"carboxyalkyl" refers to an alkyl group that is substituted with one or
more carboxy groups, "oxoalkyl" refers to an alkyl group that is
substituted with one or more oxy groups, "amino" means--NRaRb,
wherein Ra and Rb are independently selected from the group
consisting of hydrogen, alkyl and alkylcarbonyl, "amido" means
--C(O)NRaRb, wherein Ra and Rb are independently
selected from the group consisting of hydrogen and alkyl, "acyl" means
RC(O)--, "alkylcarbonyl," means an alkyl group attached to the parent
molecular moiety through a carbonyl group, "alkoxy" or "alkoxyl" means an
alkyl group, as defined herein, appended to the parent molecular moiety
through an oxygen atom, representative examples of which include, but are
not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy,
pentyloxy, and hexyloxy, "hydroxyl" means an --OH group, "carboxy" or
"carboxyl" refers to --CO2H, "halogen" means --Cl, --Br, --I or --F,
"halide" means a binary compound, of which one part is a halogen atom and
the other part is an element or radical that is less electronegative than
the halogen, e.g., an alkyl radical, "nitro" means a --NO2 group,
"sulfo" means SO3H, and "cyano" means a --CN group.

[0063]Chemiluminescent assays can be performed in accordance with the
methods described in Adamczyk et al., Anal. Chim. Acta 579(1): 61-67
(2006).

[0064]While any suitable assay format can be used, a microplate
chemiluminometer (Mithras LB-940, Berthold Technologies U.S.A., LLC, Oak
Ridge, Tenn.) enables the assay of multiple samples of small volumes
rapidly. The chemiluminometer can be equipped with multiple reagent
injectors using 96-well black polystyrene microplates (Costar #3792).
Each sample can be added into a separate well, followed by the
simultaneous/sequential addition of other reagents as determined by the
type of assay employed. Desirably, the formation of pseudobases in
neutral or basic solutions employing an acridinium aryl ester is avoided,
such as by acidification. The chemiluminescent response is then recorded
well-by-well. In this regard, the time for recording the chemiluminescent
response will depend, in part, on the delay between the addition of the
reagents and the particular acridinium employed. For example, the
emission of light from an acridinium carboxamide can be a pseudo-flash
when the reagents are added in rapid succession, such as within 5
seconds, whereas the emission of light from an acridinium carboxamide can
be a long-lived glow when there is a delay, such as 20 seconds, between
the addition of a hydrogen peroxide-generating enzyme and the acridinium
carboxamide.

[0065]The order in which the test sample and first specific binding
partner labeled with the detectable label are added to form the mixture
is not critical. After the first specific binding partner labeled with a
detectable label and the test sample are added to form the first mixture,
first specific binding partner-autoantibody complexes form.

[0066]Hydrogen peroxide can be generated in situ in the mixture or
provided or supplied to the mixture before, simultaneously with, or after
the addition of an above-described acridinium compound (specifically, the
first specific binding partner labeled with the acridinium compound).
Hydrogen peroxide can be generated in situ in a number of ways. For
example, one or more hydrogen peroxide-generating enzymes can be added to
the first mixture. The amount of one or more hydrogen peroxide-generating
enzymes to be added to the mixture can be readily determined by one
skilled in the art.

[0068]Alternatively, a source of hydrogen peroxide can be simply added to
the mixture. For example, the source of the hydrogen peroxide can be one
or more buffers or other solutions that are known to contain hydrogen
peroxide. In this regard, a solution of hydrogen peroxide can simply be
added.

[0069]Upon the addition of the acridinium, e.g., acridinium-9-carboxamide
or acridinium-9-carboxylate aryl ester, and the simultaneous or
subsequent addition of at least one basic solution to the sample, a
detectable signal, namely, a chemiluminescent signal, indicative of the
presence of autoantibody is generated. The basic solution contains at
least one base and has a pH greater than or equal to 10, preferably,
greater than or equal to 12. Examples of basic solutions include, but are
not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide,
ammonium hydroxide, magnesium hydroxide, sodium carbonate, sodium
bicarbonate, calcium hydroxide, calcium carbonate, and calcium
bicarbonate. The amount of basic solution added to the sample depends on
the concentration of the basic solution. Based on the concentration of
the basic solution used, one skilled in the art can easily determine the
amount of basic solution to add to the sample.

[0070]The chemiluminescent signal that is generated can be detected using
routine techniques known to those skilled in the art. Based on the
intensity of the signal generated, the amount of autoantibody in the
sample can be quantified. Specifically, the amount of autoantibody in the
sample is proportional to the intensity of the signal generated. The
amount of autoantibody present can be quantified by comparing the amount
of light generated to a standard curve for an anti-NGAL antibody, such as
an NGAL autoantibody, or by comparison to a reference standard. The
standard curve can be generated using serial dilutions or solutions of
known concentrations of an anti-NGAL antibody, such as an NGAL
autoantibody, by mass spectroscopy, gravimetric methods, and other
techniques known in the art.

[0071]In a heterogeneous format, after the test sample is obtained from a
subject, a first mixture is prepared. The mixture contains the test
sample being assessed for autoantibodies to NGAL (or a fragment thereof)
and a first specific binding partner, wherein the first specific binding
partner and any autoantibodies contained in the test sample form a first
specific binding partner-autoantibody complex. Preferably, the first
specific binding partner is NGAL or a fragment thereof. The order in
which the test sample and the first specific binding partner are added to
form the mixture is not critical. Preferably, the first specific binding
partner is immobilized on a solid phase. The solid phase used in the
immunoassay (for the first specific binding partner and, optionally, the
second specific binding partner) can be any solid phase known in the art,
such as, but not limited to, a magnetic particle, a bead, a test tube, a
microtiter plate, a cuvette, a membrane, a scaffolding molecule, a film,
a filter paper, a disc and a chip.

[0072]After the mixture containing the first specific binding
partner-autoantibody complex is formed, any unbound autoantibodies are
removed from the complex using any technique known in the art. For
example, the unbound autoantibodies can be removed by washing.

[0073]After any unbound autoantibodies are removed, a second specific
binding partner is added to the mixture to form a first specific binding
partner-autoantibody-second specific binding partner complex. The second
specific binding partner is preferably an anti-human antibody. Moreover,
also preferably, the second specific binding partner is labeled with or
contains a detectable label. In terms of the detectable label, any
detectable label known in the art can be used. For example, the
detectable label can be a radioactive label (such as 3H, 125I,
35S, 14C, 32P, and 33P), an enzymatic label (such as
horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate
dehydrogenase, and the like), a chemiluminescent label (such as
acridinium esters, thioesters, or sulfonamides; luminol, isoluminol,
phenanthridinium esters, and the like), a fluorescent label (such as
fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein,
3'6-carboxyfluorescein, 5(6)-carboxyfluorescein,
6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein
isothiocyanate, and the like)), rhodamine, phycobiliproteins,
R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmium
selenide), a thermometric label, or an immuno-polymerase chain reaction
label. An introduction to labels, labeling procedures and detection of
labels is found in Polak and Van Noorden, Introduction to
Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997) and in
Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996),
which is a combined handbook and catalogue published by Molecular Probes,
Inc., Eugene, Oreg. Preferably, however, the detectable label is an
acridinium compound that can be used in a chemiluminescent assay.

[0074]After the formation of the first specific binding
partner-autoantibody-second specific binding complex, any unbound second
specific binding partner (whether labeled or unlabeled) is removed from
the complex using any technique known in the art. For example, the
unbound second specific binding partner can be removed by washing.

[0075]Hydrogen peroxide can be generated in situ in the mixture or
provided or supplied to the mixture before, simultaneously with, or after
the addition of the above-described acridinium compound (specifically,
the second specific binding partner labeled with the acridinium
compound). Methods of generating hydrogen peroxide in situ are described
above.

[0076]The timing and order in which the acridinium compound (specifically,
the second specific binding partner labeled with the acridinium compound)
and the hydrogen peroxide provided in or supplied to or generated in situ
in the mixture is not critical. After the second specific binding partner
labeled with a detectable label and the test sample are added to form the
second mixture, first specific binding partner-autoantibody-second
specific binding partner complexes form.

[0077]Upon the addition of the acridinium, e.g., acridinium-9-carboxamide
or acridinium-9-carboxylate aryl ester, and the simultaneous or
subsequent addition of at least one basic solution to the sample (as
described above), a detectable signal, namely, a chemiluminescent signal,
indicative of the presence of autoantibody is generated. Chemiluminescent
signals generated can be detected using routine techniques known to those
skilled in the art.

[0078]After any unbound second specific binding partner labeled with a
detectable label is removed, a detectable signal from the detectable
label is generated or emitted and then measured. Methods for generating
signals from detectable labels and measuring the resulting signal
generated are well-known to those skilled in the art. For example, a
chemiluminescent signal can be generated after the addition of a basic
solution. The amount of the autoantibodies in the test sample can be
quantified based on the intensity of the signal generated. Specifically,
the amount of autoantibodies contained in a test sample is proportional
to the intensity of the signal generated. Specifically, the amount of
autoantibodies present can be quantified based on comparing the amount of
light generated to a standard curve for autoantibodies to a NGAL (or a
fragment thereof) or by comparison to a reference standard. The standard
curve can be generated using serial dilutions or solutions of an antibody
to NGAL (or a fragment thereof) of known concentration, by mass
spectroscopy, gravimetrically and by other techniques known in the art.

[0079]Accordingly, a method of determining the presence, amount or
concentration of at least one autoantibody that reacts with NGAL or a
fragment thereof in a test sample is provided. The method comprises
assaying the test sample for at least one autoantibody that reacts with
NGAL (or a fragment thereof). The assay employs NGAL (or a fragment
thereof) and at least one detectable label. The assay comprises comparing
a signal generated by the detectable label as a direct or indirect
indication of the presence, amount or concentration of at least one
autoantibody that reacts with NGAL (or a fragment thereof) in the test
sample to a signal generated as a direct or indirect indication of the
presence, amount or concentration of an antibody that reacts with NGAL
(or a fragment thereof) in a control or calibrator. The calibrator is
optionally part of a series of calibrators in which each of the
calibrators differs from the other calibrators in the series by the
concentration of an antibody that reacts with NGAL (or a fragment
thereof). The method can be adapted for use in an automated system or a
semi-automated system.

[0080]The method can comprise (i) contacting the test sample with NGAL (or
a fragment thereof), which comprises a detectable label and binds to at
least one autoantibody to form an NGAL (or a fragment
thereof)/autoantibody complex, and

[0081](ii) determining the presence, amount or concentration of at least
one autoantibody, which reacts with NGAL (or a fragment thereof), in the
test sample by detecting or measuring the signal generated by the
detectable label in the NGAL (or fragment thereof)/autoantibody complex
formed in (i). Preferably, the detectable label is an acridinium
compound, such as an acridinium-9-carboxamide or an
acridinium-9-carboxylate aryl ester.

[0082]The method can comprise (i) contacting the test sample with NGAL (or
a fragment thereof), which binds to at least one autoantibody and which
is optionally immobilized on a solid phase, so as to form an NGAL (or a
fragment thereof)/autoantibody complex, (ii) contacting the NGAL (or a
fragment thereof)/autoantibody complex with at least one detection
antibody, which comprises a detectable label and binds to the
autoantibody to form an NGAL (or a fragment
thereof)/autoantibody/detection antibody complex, and (iii) determining
the presence, amount or concentration of an autoantibody, which reacts
with NGAL (or fragment thereof), in the test sample by detecting or
measuring the signal generated by the detectable label in the NGAL (or a
fragment thereof)/autoantibody/detection antibody complex formed in (ii).
Optionally, the method further comprises removing any unbound at least
one autoantibody after step (i) and removing any unbound at least one
detection antibody after step (ii). Preferably, the detectable label is
an acridinium compound, such as an acridinium-9-carboxamide or an
acridinium-9-carboxylate aryl ester.

[0083]The method can further comprise determining the concentration of
NGAL (or a fragment thereof) in the test sample. The method comprises
assaying the test sample for NGAL (or a fragment thereof) by an assay
employing at least one specific binding partner for NGAL and at least one
detectable label and comprising comparing a signal generated by the
detectable label as a direct or indirect indication of the concentration
of NGAL (or a fragment thereof) in the test sample to a signal generated
as a direct or indirect indication of the concentration of NGAL in a
control or calibrator, which is optionally part of a series of
calibrators in which each of the calibrators differs from the other
calibrators in the series by the concentration of NGAL.

[0084]For example, the concentration of NGAL (or a fragment thereof) in a
test sample can be determined by an assay as described above with respect
to NGAL autoantibody or by immunoassay using any format known in the art,
such as, but not limited to, sandwich immunoassay (e.g.,
monoclonal-polyclonal sandwich immunoassays, including radioisotope
detection (radioimmunoassay (RIA)) and enzyme detection (enzyme
immunoassay (EIA) or ELISA (e.g., Quantikine ELISA assays, R&D Systems,
Minneapolis, Minn.)), competitive inhibition immunoassay (e.g., forward
and reverse), and fluorescence polarization immunoassay (FPIA). A
chemiluminescent microparticle immunoassay, in particular one employing
the ARCHITECT® automated analyzer (Abbott Laboratories, Abbott Park,
Ill.), is an example of a preferred immunoassay.

[0085]Monoclonal and polyclonal antibodies (mAbs and pAbs, respectively)
can be produced for use in immunoassays in accordance with methods known
in the art. NGAL, such as recombinantly produced NGAL, in particular,
recombinantly produced human NGAL, such as in a composition comprising an
adjuvant, can be injected into a host animal, such as a rabbit, goat,
mouse, guinea pig, or horse, at one or more sites. Further injections are
made at the same or other sites at regular or irregular intervals
thereafter with bleedings being taken to assess antibody titer until it
is determined that optimal titer has been reached. The antibodies are
obtained by either bleeding the host animal to yield a volume of
antiserum, or by somatic cell hybridization techniques or other
techniques known in the art. For example, the antibody-producing cells
can be fused by standard somatic cell fusion procedures with
immortalizing cells, such as myeloma cells, to yield hybridoma cells.
Such techniques are well-known in the art, and include, for example, the
hybridoma technique as originally developed by Kohler and Milstein,
Nature 256: 495-497 (1975)), the human B cell hybridoma technique (Kozbar
et al., Immunology Today 4: 72 (1983)), and the EBV-hybridoma technique
to produce human mAbs (Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc. pp. 77-96 (1985)). The technology for
producing mAb hybridomas is well-known to those skilled in the art (see,
e.g., Kenneth, in Monoclonal Antibodies: A New Dimension in Biological
Analyses, Plenum Pub. Corp., New York (1980)). Alternatively, anti-NGAL
antibodies can be commercially obtained from any one of a number of
sources, such as R&D Systems (Minneapolis, Minn.) among others.

[0086]In a sandwich immunoassay format, typically at least two antibodies
are used to separate and quantify an analyte of interest, in this case
NGAL (or a fragment thereof). More specifically, the two antibodies bind
to different epitopes on the analyte of interest, thereby forming what is
referred to as a "sandwich," i.e., antibody-analyte-antibody. One or more
antibodies, which bind(s) to the analyte of interest and is/are typically
bound to a substrate before or after contact with the analyte of
interest, is/are referred to as the "capture antibody" or "capture
antibodies," whereas one or more other antibodies, which is/are labeled
and bind(s) to the analyte bound by the capture antibody, is/are referred
to as the "detection antibody," "detection antibodies," "conjugate," or
"conjugates." Preferably, the binding of one antibody to the analyte does
not interfere with the binding of any other antibody to the analyte.
Also, preferably, at least the capture antibody is present in a molar
excess amount of the maximum amount of the analyte, i.e., NGAL (or
fragment thereof), expected to be present in a sample. While the
detection antibody is typically labeled prior to contact with the
analyte-capture antibody complex, the detection antibody can be labeled
simultaneously with or subsequently to the formation of the
analyte-capture antibody complex.

[0087]Generally speaking, a test sample being assayed for (for example,
suspected of containing) NGAL or a fragment thereof can be contacted with
at least one capture antibody (or antibodies) and at least one detection
antibody (which is either a second detection antibody or a third
detection antibody) either simultaneously or sequentially and in any
order. For example, the test sample can be first contacted with at least
one capture antibody and then (sequentially) with at least one detection
antibody. Alternatively, the test sample can be first contacted with at
least one detection antibody and then (sequentially) with at least one
capture antibody. In yet another alternative, the test sample can be
contacted simultaneously with a capture antibody and a detection
antibody.

[0088]In the sandwich assay format, a test sample suspected of containing
NGAL or a fragment thereof is first brought into contact with an at least
one first capture antibody under conditions, which allow the formation of
a first antibody/NGAL (or a fragment thereof) complex. If more than one
capture antibody is used, a first multiple capture antibody/NGAL (or a
fragment thereof) complex is formed. In a sandwich assay, the antibodies,
preferably, the at least one capture antibody, are used in molar excess
amounts of the maximum amount of NGAL (or a fragment thereof) expected in
the test sample. For example, from about 5 μg/mL to about 1 mg/mL of
antibody per mL of buffer (e.g., microparticle coating buffer) can be
used.

[0089]Competitive inhibition immunoassays, which are often used to measure
small analytes because binding by only one antibody is required, comprise
sequential and classic formats. In a sequential competitive inhibition
immunoassay a capture mAb to an analyte of interest is coated onto a well
of a microtiter plate. When the sample containing the analyte of interest
is added to the well, the analyte of interest binds to the capture mAb.
After washing, a known amount of labeled (e.g., biotin or horseradish
peroxidase (HRP)) analyte is added to the well. A substrate for an
enzymatic label is necessary to generate a signal. An example of a
suitable substrate for HRP is 3,3',5,5'-tetramethylbenzidine (TMB). After
washing, the signal generated by the labeled analyte is measured and is
inversely proportional to the amount of analyte in the sample. In a
classic competitive inhibition immunoassay an mAb to an analyte of
interest is coated onto a well of a microtiter plate. However, unlike the
sequential competitive inhibition immunoassay, the sample and the labeled
analyte are added to the well at the same. Any analyte in the sample
competes with labeled analyte for binding to the capture mAb. After
washing, the signal generated by the labeled analyte is measured and is
inversely proportional to the amount of analyte in the sample.

[0090]Optionally, prior to contacting the test sample with the at least
one capture antibody (for example, the first capture antibody), the at
least one capture antibody can be bound to a solid support, which
facilitates the separation of the first antibody/NGAL (or a fragment
thereof) complex from the test sample. The substrate to which the capture
antibody is bound can be any suitable solid support or solid phase that
facilitates separation of the capture antibody-analyte complex from the
sample. Examples include a well of a plate, such as a microtiter plate, a
test tube, a porous gel (e.g., silica gel, agarose, dextran, or gelatin),
a polymeric film (e.g., polyacrylamide), beads (e.g., polystyrene beads
or magnetic beads), a strip of a filter/membrane (e.g., nitrocellulose or
nylon), microparticles (e.g., latex particles, magnetizable
microparticles (e.g., microparticles having ferric oxide or chromium
oxide cores and homo- or hetero-polymeric coats and radii of about 1-10
microns), sheep red blood cells, or DURACYTES® (Abbott Laboratories),
which are red blood cells that have been "fixed" by pyruvic aldehyde and
formaldehyde)). The substrate can comprise a suitable porous material
with a suitable surface affinity to bind antigens and sufficient porosity
to allow access by detection antibodies. A microporous material is
generally preferred, although a gelatinous material in a hydrated state
can be used. Such porous substrates are preferably in the form of sheets
having a thickness of about 0.01 to about 0.5 mm, preferably about 0.1
mm. While the pore size may vary quite a bit, preferably the pore size is
from about 0.025 to about 15 microns, more preferably from about 0.15 to
about 15 microns. The surface of such substrates can be activated by
chemical processes that cause covalent linkage of an antibody to the
substrate. Irreversible binding, generally by adsorption through
hydrophobic forces, of the antigen or the antibody to the substrate
results; alternatively, a chemical coupling agent or other means can be
used to bind covalently the antibody to the substrate, provided that such
binding does not interfere with the ability of the antibody to bind
IL-18. Alternatively, the antibody can be bound with microparticles,
which have been previously coated with streptavidin or biotin (e.g.,
using Power-Bind®-SA-MP streptavidin-coated microparticles (Seradyn,
Indianapolis, Ind.)) or anti-species-specific mAbs. If necessary, the
substrate can be derivatized to allow reactivity with various functional
groups on the antibody. Such derivatization requires the use of certain
coupling agents, examples of which include, but are not limited to,
maleic anhydride, N-hydroxysuccinimide, and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0091]After the test sample being assayed for NGAL (or a fragment thereof)
is brought into contact with the at least one capture antibody (for
example, the first capture antibody), the mixture is incubated in order
to allow for the formation of a first antibody (or multiple
antibody)-NGAL (or a fragment thereof) complex. The incubation can be
carried out at a pH of from about 4.5 to about 10.0, at a temperature of
from about 2° C. to about 45° C., and for a period from at
least about one (1) minute to about eighteen (18) hours, preferably from
about 1 to about 24 minutes, most preferably for about 4 to about 18
minutes. The immunoassay described herein can be conducted in one step
(meaning the test sample, at least one capture antibody and at least one
detection antibody are all added sequentially or simultaneously to a
reaction vessel) or in more than one step, such as two steps, three
steps, etc.

[0092]After formation of the (first or multiple) capture antibody/NGAL (or
a fragment thereof) complex, the complex is then contacted with at least
one detection antibody (under conditions which allow for the formation of
a (first or multiple) capture antibody/NGAL (or a fragment
thereof)/second antibody detection complex). The at least one detection
antibody can be the second, third, fourth, etc. antibodies used in the
immunoassay. If the capture antibody/NGAL (or a fragment thereof) complex
is contacted with more than one detection antibody, then a (first or
multiple) capture antibody/NGAL (or a fragment thereof)/(multiple)
detection antibody complex is formed. As with the capture antibody (e.g.,
the first capture antibody), when the at least second (and subsequent)
detection antibody is brought into contact with the capture antibody/NGAL
(or a fragment thereof) complex, a period of incubation under conditions
similar to those described above is required for the formation of the
(first or multiple) capture antibody/NGAL (or a fragment thereof)/(second
or multiple) detection antibody complex. Preferably, at least one
detection antibody contains a detectable label. The detectable label can
be bound to the at least one detection antibody (e.g., the second
detection antibody) prior to, simultaneously with, or after the formation
of the (first or multiple) capture antibody/NGAL (or a fragment
thereof)/(second or multiple) detection antibody complex. Any detectable
label known in the art can be used (see discussion above, including Polak
and Van Noorden (1997) and Haugland (1996)).

[0093]The detectable label can be bound to the antibodies either directly
or through a coupling agent. An example of a coupling agent that can be
used is EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide,
hydrochloride), which is commercially available from Sigma-Aldrich, St.
Louis, Mo. Other coupling agents that can be used are known in the art.
Methods for binding a detectable label to an antibody are known in the
art. Additionally, many detectable labels can be purchased or synthesized
that already contain end groups that facilitate the coupling of the
detectable label to the antibody, such as CPSP-Acridinium Ester or
SPSP-Acridinium Ester.

[0094]The (first or multiple) capture antibody/NGAL (or a fragment
thereof)/(second or multiple) detection antibody complex can be, but does
not have to be, separated from the remainder of the test sample prior to
quantification of the label. For example, if the at least one capture
antibody (e.g., the first capture antibody) is bound to a solid support,
such as a well or a bead, separation can be accomplished by removing the
fluid (of the test sample) from contact with the solid support.
Alternatively, if the at least first capture antibody is bound to a solid
support, it can be simultaneously contacted with the NGAL (or a fragment
thereof)-containing sample and the at least one second detection antibody
to form a first (multiple) antibody/NGAL (or a fragment thereof)/second
(multiple) antibody complex, followed by removal of the fluid (test
sample) from contact with the solid support. If the at least one first
capture antibody is not bound to a solid support, then the (first or
multiple) capture antibody/NGAL (or a fragment thereof)/(second or
multiple) detection antibody complex does not have to be removed from the
test sample for quantification of the amount of the label.

[0095]After formation of the labeled capture antibody/NGAL (or a fragment
thereof)/detection antibody complex (e.g., the first capture
antibody/NGAL (or a fragment thereof)/second detection antibody complex),
the amount of label in the complex is quantified using techniques known
in the art. For example, if an enzymatic label is used, the labeled
complex is reacted with a substrate for the label that gives a
quantifiable reaction, such as the development of color. If the label is
a radioactive label, the label is quantified using a scintillation
counter. If the label is a fluorescent label, the label is quantified by
stimulating the label with a light of one color (which is known as the
"excitation wavelength") and detecting another color (which is known as
the "emission wavelength") that is emitted by the label in response to
the stimulation. If the label is a chemiluminescent label, the label is
quantified detecting the light emitted either visually or by using
luminometers, x-ray film, high-speed photographic film, a CCD camera,
etc. Once the amount of the label in the complex has been quantified, the
concentration of NGAL (or a fragment thereof) in the test sample is
determined by use of a standard curve that has been generated using
serial dilutions of NGAL (or a fragment thereof) of known concentration.
Other than using serial dilutions of NGAL (or a fragment thereof), the
standard curve can be generated gravimetrically, by mass spectroscopy and
by other techniques known in the art.

[0096]The NGAL assay can employ a monoclonal antibody (mAb) sandwich that
utilizes a capture antibody that binds only free NGAL and excludes bound
NGAL, such as NGAL bound to metalloproteinase-9 (MMP-9) or gelatinase B.
The amount of captured free NGAL can be detected with an acridinylated
anti-NGAL mAb.

[0097]FPIAs are based on competitive binding immunoassay principles. A
fluorescently labeled compound, when excited by a linearly polarized
light, will emit fluorescence having a degree of polarization inversely
proportional to its rate of rotation. When a fluorescently labeled
tracer-antibody complex is excited by a linearly polarized light, the
emitted light remains highly polarized because the fluorophore is
constrained from rotating between the time light is absorbed and the time
light is emitted. When a "free" tracer compound (I.e., a compound that is
not bound to an antibody) is excited by linearly polarized light, its
rotation is much faster than the corresponding tracer-antibody conjugate
produced in a competitive binding immunoassay. FPIAs are advantageous
over RIAs inasmuch as there are no radioactive substances requiring
special handling and disposal. In addition, FPIAs are homogeneous assays
that can be easily and rapidly performed.

[0098]The method can further comprise diagnosing, prognosticating, or
assessing the efficacy of a therapeutic/prophylactic treatment of a
patient from whom the test sample was obtained. If the method further
comprises assessing the efficacy of a therapeutic/prophylactic treatment
of the patient from whom the test sample was obtained, the method
optionally further comprises modifying the therapeutic/prophylactic
treatment of the patient as needed to improve efficacy.

[0099]The methods described herein can be used for determining the
reliability of a result obtained from an assay (e.g., namely, an assay
that was previously, simultaneously or subsequently performed) for
detecting or quantifying the amount of NGAL in a test sample obtained
from a subject. Specifically, such a method involves obtaining a test
sample (a single test sample can be obtained from a subject and split
between the autoantibody assay and the NGAL assay) from the same subject
and determining the presence or the concentration of at least one
autoantibody that reacts with NGAL using any of the assays described
herein or any alternate assay, which may or may not be an immunoassay,
for an autoantibody as known in the art. If the concentration of the at
least one autoantibody is elevated compared to a predetermined level,
then the concentration of NGAL as determined by separate assay is
considered not to be reliable. However, if the concentration of the at
least one autoantibody is lower or the same as a predetermined level,
then the concentration of NGAL as determined by separate assay is
considered to be reliable.

[0100]Generally, a predetermined level can be employed as a benchmark
against which to assess results obtained upon assaying a test sample for
at least one autoantibody that reacts with NGAL or a fragment thereof.
Generally, in making such a comparison, the predetermined level is
obtained by running a particular assay a sufficient number of times and
under appropriate conditions such that a linkage or association of
analyte (e.g., autoantibody) presence, amount or concentration with a
particular stage or endpoint of a disease, disorder or condition (e.g.,
cardiovascular disease or renal disease) or with particular clinical
indicia can be made. Typically, the predetermined level is obtained with
assays of reference subjects (or populations of subjects).

[0101]With respect to at least one autoantibody reactive with NGAL (or a
fragment thereof), it is envisioned that such autoantibodies can be
directed against a variety of in vivo targets associated with the
cardiovascular system or other major organ systems, such as the excretory
system (e.g., the kidneys), in which NGAL has any function or impact.
Accordingly, a particular autoantibody may either or increase or decrease
with respect to a predetermined level.

[0102]In particular, with respect to a predetermined level as employed for
monitoring disease progression and/or treatment, the concentration or
amount of an autoantibody reactive with NGAL or fragment thereof may be
either "unchanged," "favorable" (or "favorably altered"), or
"unfavorable" (or "unfavorably altered"). Generally, because
autoantibodies as described herein appear to be associated with an
NGAL-specific cardiophysiopathology and are elevated in a low proportion
(e.g., less than about five %, especially from about 0.5% to about 5%) of
the so-called normal population and are elevated in a higher proportion
(less than about 15%, especially from about 10 to about 15%) of the
population testing positive for the presence of NGAL, it is likely in
most cases that "unfavorable" ("unfavorably altered") corresponds to an
increase or elevation in autoantibody amount or concentration, and
"favorable" ("favorably altered") corresponds to an decrease or reduction
in autoantibody amount or concentration, in each case relative to a
predetermined level or to a prior measured value.

[0103]As used herein, the term "elevated" or "increased" refers to a
concentration or amount in a test sample that is higher than a typical or
normal level or range (e.g., predetermined level), or is higher that
another reference level or range (e.g., earlier or baseline sample). The
term "lowered" or "reduced" refers to a concentration or amount in a test
sample that is higher than a typical or normal level or range (e.g.,
predetermined level), or is higher that another reference level or range
(e.g., earlier or baseline sample). The term "altered" refers to a
concentration or amount in a sample that is altered (increased or
decreased) over a typical or normal level or range (e.g., predetermined
level), or over another reference level or range (e.g., earlier or
baseline sample).

[0104]The typical or normal level or range for NGAL and autoantibodies
reactive therewith is defined in accordance with standard practice.
Because the levels of autoantibodies in some instances will be very low,
a so-called altered level or alteration can be considered to have
occurred when there is any net change as compared to the typical or
normal level or range, or reference level or range, that cannot be
explained by experimental error or sample variation. Thus, the level
measured in a particular sample will be compared with the level or range
of levels determined in similar samples from a so-called normal subject.
In this context, a "normal subject" is an individual with no detectable
cardiovascular or renal pathology, for example, and a "normal" (sometimes
termed "control") patient or population is/are one(s) that exhibits no
detectable cardiovascular or renal pathology, for example. Furthermore,
given that one or more autoantibodies against NGAL are not routinely
found at high levels in the majority of the human population, a "normal
subject" can be considered an individual with no substantial detectable
increased or elevated concentration or amount of NGAL autoantibodies, and
a "normal" (sometimes termed "control") patient or population is/are
one(s) that exhibits no substantial detectable increased or elevated
concentration or amount of NGAL autoantibodies. An "apparently normal
subject" is one in which autoantibodies have not been or are being
assessed. The level of an analyte is said to be "elevated" when the
analyte is normally undetectable (e.g., the normal level is zero, or
within a range of from about 25 to about 75 percentiles of normal
populations), but is detected in a test sample, as well as when the
analyte is present in the test sample at a higher than normal level.

[0105]As previously mentioned, it is believed that one or more
autoantibodies reactive with NGAL or a fragment thereof as described
herein can be directed against a variety of in vivo targets associated
with the cardiovascular system or other major organ systems (e.g.,
excretory system, in particular the kidneys) in which NGAL has any
function or impact. Thus, inter alia, the disclosure provides a method of
screening for a subject having, or at risk of having, cardiovascular
disease or renal disease, for example, as defined herein.

[0106]Any of the test methods as described herein can be performed in
conjunction with one or more other tests including, but not limited to,
physical examination, and/or the taking of a medical history to allow a
differential diagnosis of cardiovascular or renal disease. The various
tests and parameters employed in diagnosing these disorders are
well-known to those of skill in the art. Furthermore, any of the methods
can be carried out on samples from asymptomatic subjects or subjects
having one or more risk factors associated with, or symptoms of,
cardiovascular disease or renal disease.

[0107]In particular embodiments, when a subject is determined to have an
unfavorable level of one or more autoantibodies to NGAL, the subject
optionally is assessed for one or more additional indicators of
cardiovascular disease, such as myoglobin, CK-MB (creatine kinase
muscle-brain), BNP (brain natriuretic peptide), CRP (C reactive protein),
cardiac troponin I (cTnI), cardiac troponin T (cTnT), blood oxygen level,
cardiac imaging, electrocardiography, and any others that are known in
the art. Alternatively or additionally, the subject optionally is
assessed for one or more additional indicators of renal disease, such as
proteinuria, heamaturia, serum creatine, cystatin C,
S-adenosylhomocysteine, homocysteine, an abnormally high body mass index
(BMI), obesity, and others as known in the art.

[0109]Thus, in one embodiment provided herein is a method for determining
the reliability of a result obtained from a separate assay that was
previously, simultaneously or subsequently performed for detecting or
quantifying the amount or concentration of NGAL in a test sample obtained
from a subject, wherein the method comprises:

[0110](a) determining the amount or the concentration in the test sample
of at least one autoantibody that reacts with NGAL; and

[0111](b) comparing the amount or the concentration in (a) to a
predetermined level, wherein if the amount or the concentration is
elevated as compared to a predetermined level, then the amount or
concentration of NGAL as determined by separate assay is considered not
to be reliable, and wherein if the amount or the concentration in (a) is
lower or the same as a predetermined level, then the amount or
concentration of NGAL as determined by separate assay is considered to be
reliable.

[0112]Accordingly, the methods described herein also can be used to
determine whether or not a subject has or is at risk of developing a
cardiovascular disease or a renal disease. Specifically, such a method
can comprise the steps of:

[0113](a) determining the concentration or amount in a test sample from a
subject of at least one autoantibody that reacts with NGAL (or a fragment
thereof) (e.g., using the methods described herein, or methods known in
the art); and

[0114](b) comparing the concentration or amount of at least one
autoantibody that reacts with NGAL (or fragment thereof) determined in
step (a) with a predetermined level, wherein, if the concentration or
amount of the one or more autoantibodies reactive with human NGAL
determined in step (a) is favorable with respect to a predetermined
level, then the subject is determined not to have or be at risk for a
cardiovascular disease or renal disease. However, if the concentration or
amount of the one or more autoantibodies reactive with NGAL determined in
step (a) is unfavorable with respect to the predetermined level, then the
subject is determined to have or be at risk for a cardiovascular disease
or renal disease.

[0115]Additionally, provided herein is method of monitoring the
progression of disease in a subject. Optimally the method comprising the
steps of:

[0116](a) determining the concentration or amount in a test sample from a
subject of at least one autoantibody reactive with NGAL;

[0117](b) determining the concentration or amount in a later test sample
from the subject of at least one autoantibody reactive with NGAL; and

[0118](c) comparing the concentration or amount of at least one
autoantibody reactive with NGAL as determined in step (b) with the
concentration or amount of at least one autoantibody reactive with NGAL
determined in step (a), wherein if the concentration or amount determined
in step (b) is unchanged or is unfavorable when compared to the
concentration or amount of at least one autoantibody reactive with NGAL
determined in step (a), then the disease in the subject is determined to
have continued, progressed or worsened. By comparison, if the
concentration or amount of at least one autoantibody reactive with NGAL
determined in step (b) is favorable when compared to the concentration or
amount of at least one autoantibody reactive with NGAL as determined in
step (a), then the disease in the subject is determined to have
discontinued, regressed or improved.

[0119]Optionally, the method further comprises comparing the concentration
or amount of at least one autoantibody reactive with NGAL determined in
step (b), for example, with a predetermined level. Further, optionally
the method comprises treating the subject with one or more pharmaceutical
compositions for a period of time if the comparison shows that the
concentration or amount of at least one autoantibody reactive with NGAL
as determined in step (b), for example, is unfavorably altered with
respect to the predetermined level.

[0120]Still further, the methods can be used to monitor treatment in a
subject receiving treatment with one or more pharmaceutical compositions.
Specifically, such methods involve providing a first test sample from a
subject before the subject has been administered one or more
pharmaceutical compositions. Next, the concentration or amount in a first
test sample from a subject of at least one autoantibody reactive with
NGAL is determined (e.g., using the methods described herein or as known
in the art). After the concentration or amount of at least one
autoantibody reactive with NGAL is determined, optionally the
concentration or amount of at least one autoantibody reactive with NGAL
is then compared with a predetermined level. If the concentration or
amount of the at least one autoantibody reactive with NGAL as determined
in the first test sample is lower than the predetermined level, then the
subject is not treated with one or more pharmaceutical compositions.
However, if the concentration or amount of the at least one autoantibody
reactive with NGAL as determined in the first test sample is higher than
the predetermined level, then the subject is treated with one or more
pharmaceutical compositions for a period of time. The period of time that
the subject is treated with the one or more pharmaceutical compositions
can be determined by one skilled in the art (for example, the period of
time can be from about seven (7) days to about two years, preferably from
about fourteen (14) days to about one (1) year).

[0121]During the course of treatment with the one or more pharmaceutical
compositions, second and subsequent test samples are then obtained from
the subject. The number of test samples and the time in which said test
samples are obtained from the subject are not critical. For example, a
second test sample could be obtained seven (7) days after the subject is
first administered the one or more pharmaceutical compositions, a third
test sample could be obtained two (2) weeks after the subject is first
administered the one or more pharmaceutical compositions, a fourth test
sample could be obtained three (3) weeks after the subject is first
administered the one or more pharmaceutical compositions, a fifth test
sample could be obtained four (4) weeks after the subject is first
administered the one or more pharmaceutical compositions, etc.

[0122]After each second or subsequent test sample is obtained from the
subject, the concentration or amount of at least one autoantibody
reactive with NGAL is determined in the second or subsequent test sample
is determined (e.g., using the methods described herein or as known in
the art). The concentration or amount of at least one autoantibody
reactive with NGAL as determined in each of the second and subsequent
test samples is then compared with the concentration or amount of at
least one autoantibody reactive with NGAL as determined in the first test
sample (e.g., the test sample that was originally optionally compared to
the predetermined level). If the concentration or amount of at least one
autoantibody reactive with NGAL as determined in step (c) is favorable
when compared to the concentration or amount of at least one autoantibody
reactive with NGAL as determined in step (a), then the disease in the
subject is determined to have discontinued, regressed or improved, and
the subject should continue to be administered the one or pharmaceutical
compositions of step (b). However, if the concentration or amount
determined in step (c) is unchanged or is unfavorable when compared to
the concentration or amount of at least one autoantibody reactive with
NGAL determined in step (a), then the disease in the subject is
determined to have continued, progressed or worsened, and the subject
should be treated with a higher concentration of the one or more
pharmaceutical compositions administered to the subject in step (b) or
the subject should be treated with one or more pharmaceutical
compositions that are different from the one or more pharmaceutical
compositions administered to the subject in step (b). Specifically, the
subject can be treated with one or more pharmaceutical compositions that
are different from the one or more pharmaceutical compositions that the
subject had previously received to decrease or lower said subject's NGAL
autoantibodies levels.

[0123]Generally, for assays in which repeat testing may be done (e.g.,
monitoring disease progression and/or response to treatment), a second or
subsequent test sample is obtained at a period in time after the first
test sample has been obtained from the subject. Specifically, a second
test sample from the subject can be obtained minutes, hours, days, weeks
or years after the first test sample has been obtained from the subject.
For example, the second test sample can be obtained from the subject at a
time period of about 1 minute, about 5 minutes, about 10 minutes, about
15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2
hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about
7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours,
about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16
hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,
about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2
days, about 3 days, about 4 days, about 5 days, about 6 days, about 7
days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6
weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about
11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks,
about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20
weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks,
about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29
weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks,
about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38
weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks,
about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47
weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks,
about 52 weeks, about 1.5 years, about 2 years, about 2.5 years, about
3.0 years, about 3.5 years, about 4.0 years, about 4.5 years, about 5.0
years, about 5.5 years, about 6.0 years, about 6.5 years, about 7.0
years, about 7.5 years, about 8.0 years, about 8.5 years, about 9.0
years, about 9.5 years or about 10.0 years after the first test sample
from the subject is obtained. When used to monitor disease progression,
the above assay can be used to monitor the progression of disease in
subjects suffering from acute conditions. Acute conditions, also known as
critical care conditions, refer to acute, life-threatening diseases or
other critical medical conditions involving, for example, the
cardiovascular system or excretory system. Typically, critical care
conditions refer to those conditions requiring acute medical intervention
in a hospital-based setting (including, but not limited to, the emergency
room, intensive care unit, trauma center, or other emergent care setting)
or administration by a paramedic or other field-based medical personnel.
For critical care conditions, repeat monitoring is generally done within
a shorter time frame, namely, minutes, hours or days (e.g., about 1
minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30
minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3
hours, about 4 hours, 4 about 5 hours, about 6 hours, about 7 hours,
about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12
hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours,
about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21
hours, about 22 hours, about 23 hours, about 24 hours, about 2 days,
about 3 days, about 4 days, about 5 days, about 6 days or about 7 days),
and the initial assay likewise is generally done within a shorter
timeframe, e.g., about minutes, hours or days of the onset of the disease
or condition.

[0124]The assays also can be used to monitor the progression of disease in
subjects suffering from chronic or non-acute conditions. Non-critical
care or, non-acute conditions, refers to conditions other than acute,
life-threatening disease or other critical medical conditions involving,
for example, the cardiovascular system and/or excretory system.
Typically, non-acute conditions include those of longer-term or chronic
duration. For non-acute conditions, repeat monitoring generally is done
with a longer timeframe, e.g., hours, days, weeks, months or years (e.g.,
about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours,
about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10
hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours,
about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19
hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours,
about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days,
about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks,
about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9
weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks,
about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18
weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks,
about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27
weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks,
about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36
weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks,
about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45
weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks,
about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2
years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0
years, about 4.5 years, about 5.0 years, about 5.5 years, about 6.0
years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0
years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0
years), and the initial assay likewise generally is done within a longer
time frame, e.g., about hours, days, months or years of the onset of the
disease or condition.

[0125]Furthermore, the above assays can be performed using a first test
sample obtained from a subject where the first test sample is whole
blood, serum, or plasma. The above assays can then be repeated using a
second test sample obtained from the subject where the second test sample
is something other than whole blood, serum or plasma (e.g., urine). The
results obtained from the assays using the first test sample and the
second test sample can be compared. The comparison can be used to assess
the status of a disease or condition in the subject.

[0126]Moreover, the present disclosure also relates to methods of
determining whether a subject predisposed to or suffering from a disease
(e.g., cardiovascular disease or renal disease) will benefit from
treatment. In particular, the disclosure relates to NGAL companion
diagnostic methods and products. Thus, the method of "monitoring the
treatment of disease in a subject" as described herein further optimally
also can encompass selecting or identifying candidates for therapy.

[0127]Thus, in particular embodiments, the disclosure also provides a
method of determining whether a subject having, or at risk for,
cardiovascular or renal disease is a candidate for therapy. Generally,
the subject is one who has experienced some symptom of cardiovascular
disease or who has actually been diagnosed as having, or being at risk
for, cardiovascular or renal disease, and/or who demonstrates an
unfavorable concentration or amount of at least one autoantibody reactive
with NGAL or a fragment thereof, as described herein.

[0128]The method optionally comprises an assay as described herein, where
analyte is assessed before and following treatment of a subject with one
or more pharmaceutical compositions (e.g., particularly with a
pharmaceutical related to a mechanism of action involving NGAL), with
immunosuppressive therapy, or by immunoabsorption therapy, or where
analyte is assessed following such treatment and the concentration or the
amount of analyte is compared against a predetermined level. An
unfavorable concentration of amount of analyte observed following
treatment confirms that the subject will not benefit from receiving
further or continued treatment, whereas a favorable concentration or
amount of analyte observed following treatment confirms that the subject
will benefit from receiving further or continued treatment. This
confirmation assists with management of clinical studies, and provision
of improved patient care.

[0129]It goes without saying that while certain embodiments herein are
advantageous when employed to assess cardiovascular or renal disease, the
assays and kits also optionally can be employed to assess NGAL
autoantibodies in other diseases, disorders and conditions, e.g., cancer,
sepsis, and any disease, disorder or condition that might involve an
assessment of NGAL or autoantibody thereto.

[0131]Moreover, any of the teachings of U.S. Provisional Application Nos.
60/981,470, 60/981,471 and 60/981,473, all filed on Oct. 19, 2007, and
U.S. patent application Ser. Nos. 12/104,408, 12/104,410, and 12/104,413,
all filed on Apr. 16, 2008, with respect to assay rare reagents NGAL
antigen, anti-NGAL antibody, and an NGAL assay can be applied in the
methods and kits as described herein and are each incorporated by
reference in their entireties for their teachings regarding same.

Isolated Autoantibodies Reactive with Human NGAL or a Fragment Thereof

[0132]Also provided are isolated human NGAL autoantibodies. The isolated
human NGAL autoantibodies can be IgG, IgA, or IgM antibodies. Preferably,
the isolated autoantibodies are IgG antibodies. The autoantibodies can be
obtained using routine techniques known in the art. For example, the
autoantibodies can be obtained by separating such autoantibodies from
their environment, such as, for example, from a mixture containing human
NGAL autoantibodies. Such a mixture can be obtained from one or more
subjects during the course of normal blood donation. Further, such a
mixture can be obtained from one or more subjects receiving treatment
with a human NGAL (or fragment or derivative thereof). Alternatively, the
mixture can be obtained from one or more subjects with clinical signs of
cardiovascular or renal disease or an endogenous concentration of NGAL
that is higher than the clinically acceptable value for a normal
population. Mixtures containing such autoantibodies can be obtained from
freshly collected or stored blood, plasma or serum from such a subject
and readily identified using the methods described herein. The
autoantibodies can be isolated using routine immunoglobulin procedures,
such as salt fractionation (e.g., ammonium sulfate precipitation),
immunoprecipitation, affinity capture on a solid phase, gel
electrophoresis, dialysis, or chromatography (e.g., protein A-sepharose
chromatography, hydroxyapatite chromatography, affinity chromatography,
anion-exchange chromatography, ion-exchange chromatography, immunoffinity
chromatography, size exclusion chromatography, reversed-phase
chromatography, etc.).

[0134]Once the amino acid sequence of the autoantibody is determined, the
nucleic acid sequence of the autoantibody also can be determined using
routine techniques known in the art. The nucleic acid sequence thus
obtained can be used to express the autoantibody or autoantibody
fragments in human or non-human cell lines, or non-human subjects as
well-known to those skilled in the art of antibody engineering.

[0135]Additionally, once the amino acid and nucleic acid sequences of the
isolated NGAL autoantibody are obtained, the amino acid and nucleic acid
sequences can be directly synthesized using various solid-phase
techniques (see, e.g., Roberge et al., Science 269: 202-204 (1995)), and
automated synthesis can be achieved, for example, using the ABI 43 1 A
Peptide Synthesizer (Perkin Elmer) in accordance with the instructions
provided by the manufacturer. Additionally, the amino acid sequence
obtainable from the isolated autoantibody described herein can be altered
during direct synthesis and/or combined using chemical methods with a
sequence from other subunits, or any part thereof, to produce variant
sequences and hence variant human NGAL autoantibodies.

[0136]The human NGAL autoantibodies described herein can be used for a
variety of different purposes. For example, these isolated autoantibodies
can be used in screening methods to identify agents that are useful in
inhibiting the binding of human NGAL or fragment thereof to at least one
human anti-NGAL autoantibody. Specifically, such screening methods would
involve preparing a mixture comprising an isolated human NGAL
autoantibody. After such a mixture is prepared, the method would involve
adding to the mixture, either simultaneously or sequentially, and in any
order, human NGAL or fragment thereof and at least one agent to be tested
(such as a pharmaceutical composition, etc.). The final step would
involve determining whether the agent being tested inhibits the binding
of NGAL or fragment thereof to the human NGAL autoantibody. It is
contemplated that such a method could be partially or fully automated to
allow for the screening of a large number of agents at one time. Agents
determined to inhibit the binding of NGAL or a fragment thereof to the
human NGAL autoantibody would be selected for further study for use as a
potential therapeutic agent in treating cardiovascular or renal disease
in a human. Additionally, the isolated autoantibodies described herein
also could be employed in pharmaceutical compositions that can be used to
treat cardiovascular or renal disease. Such pharmaceutical compositions
would contain the isolated autoantibodies described herein and one or
more pharmaceutically acceptable excipients.

[0137]In view of the above, a method of isolating an autoantibody that
reacts with NGAL or a fragment thereof is provided. The method comprises
the steps of (i) contacting NGAL (or a fragment thereof) with a
biological sample, which is known to contain an autoantibody that reacts
with NGAL, wherein the NGAL is optionally immobilized on a solid phase
before or after contact with the biological sample, (ii) isolating NGAL
to which is bound the autoantibody, and (iii) isolating the autoantibody
from the NGAL. Also provided is an isolated autoantibody that reacts with
NGAL or a fragment thereof.

Assay Kit

[0138]A kit for assaying a test sample for the presence, amount or
concentration of at least one autoantibody that reacts with NGAL (or a
fragment thereof) in a test sample is also provided. The kit comprises at
least one component for assaying the test sample for at least one
autoantibody that reacts with NGAL (or a fragment thereof) and
instructions for assaying the test sample for at least one autoantibody
that reacts with NGAL (or a fragment thereof). The at least one component
for assaying the test sample for at least one autoantibody that reacts
with NGAL (or a fragment thereof) includes a composition comprising NGAL
(or a fragment thereof), which is optionally immobilized on a solid
phase, and/or a composition comprising an antibody that can bind to the
at least one autoantibody that reacts with NGAL (or a fragment thereof),
wherein the antibody is optionally detectably labeled. The instructions
can also contain instructions for generating a standard curve or a
reference standard for purposes of quantifying the autoantibodies. Such
instructions optionally can be in printed form or on CD, DVD, or other
format of recorded media.

[0139]The kit can further comprise at least one component for assaying the
test sample for NGAL (or a fragment thereof) and instructions for
assaying the test sample for NGAL (or a fragment thereof). The at least
one component for assaying the test sample for NGAL (or a fragment
thereof) includes a composition comprising an antibody that can bind to
NGAL (or a fragment thereof), wherein the antibody is optionally
detectably labeled.

[0140]Alternatively or additionally, the kit can comprise a calibrator or
control, e.g., isolated or purified, NGAL antibody, and/or at least one
container (e.g., tube, microtiter plates or strips, which can be already
coated with NGAL) for conducting the assay, and/or a buffer, such as an
assay buffer or a wash buffer, either one of which can be provided as a
concentrated solution, a substrate solution for the detectable label
(e.g., an enzymatic label), or a stop solution. Preferably, the kit
comprises all components, i.e., reagents, standards, buffers, diluents,
etc., which are necessary to perform the assay.

[0141]Any antibodies, which are provided in the kit, such as antibodies
specific for NGAL, can incorporate a detectable label, such as a
fluorophore, radioactive moiety, enzyme, biotin/avidin label,
chromophore, chemiluminescent label, or the like, or the kit may include
reagents for labeling the antibodies or reagents for detecting the
antibodies (e.g., detection antibodies) and/or for labeling the analytes
or reagents for detecting the analyte. The antibodies, calibrators and/or
controls can be provided in separate containers or pre-dispensed into an
appropriate assay format, for example, into microtiter plates.

[0142]Optionally, the kit includes quality control components (for
example, sensitivity panels, calibrators, and positive controls).
Preparation of quality control reagents is well-known in the art and is
described on insert sheets for a variety of immunodiagnostic products.
Sensitivity panel members optionally are used to establish assay
performance characteristics, and further optionally are useful indicators
of the integrity of the immunoassay kit reagents, and the standardization
of assays.

[0143]The kit can also optionally include other reagents required to
conduct a diagnostic assay or facilitate quality control evaluations,
such as buffers, salts, enzymes, enzyme co-factors, substrates, detection
reagents, and the like. Other components, such as buffers and solutions
for the isolation and/or treatment of a test sample (e.g., pretreatment
reagents), also can be included in the kit. The kit can additionally
include one or more other controls. One or more of the components of the
kit can be lyophilized, in which case the kit can further comprise
reagents suitable for the reconstitution of the lyophilized components.

[0144]The various components of the kit optionally are provided in
suitable containers as necessary, e.g., a microtiter plate. The kit can
further include containers for holding or storing a sample (e.g., a
container or cartridge for a urine sample). Where appropriate, the kit
optionally also can contain reaction vessels, mixing vessels, and other
components that facilitate the preparation of reagents or the test
sample. The kit can also include one or more instrument for assisting
with obtaining a test sample, such as a syringe, pipette, forceps,
measured spoon, or the like.

[0145]Preferably, the detectable label is at least one acridinium compound
as described herein. The kit can comprise at least one
acridinium-9-carboxamide, at least one acridinium-9-carboxylate aryl
ester, or any combinations thereof. If the detectable label is at least
one acridinium compound, the kit also can comprise a source of hydrogen
peroxide, such as a buffer, solution, a composition comprising at least
one hydrogen peroxide-generating enzyme, or another means of generating
hydrogen peroxide in situ, and/or at least one basic solution. If
desired, the kit can contain a solid phase, such as a magnetic particle,
bead, test tube, microtiter plate, cuvette, membrane, scaffolding
molecule, film, filter paper, disc or chip.

Adaptation of Method and Assay Kit

[0146]The kit (or components thereof), as well as the method of
determining the concentration of NGAL autoantibody in a test sample by an
assay as described below, can be adapted for use in a variety of
automated and semi-automated systems (including those wherein the solid
phase comprises a microparticle), as described, e.g., in U.S. Pat. Nos.
5,089,424 and 5,006,309, and as commercially marketed, e.g., by Abbott
Laboratories (Abbott Park, Ill.) as ARCHITECT®.

[0147]Some of the differences between an automated or semi-automated
system as compared to a non-automated system (e.g., ELISA) include the
substrate to which the first specific binding partner (e.g., NGAL) is
attached (which can impact sandwich formation and analyte reactivity),
and the length and timing of the capture, detection and/or any optional
wash steps. Whereas a non-automated format such as an ELISA may require a
relatively longer incubation time with sample and capture reagent (e.g.,
about 2 hours) an automated or semi-automated format (e.g.,
ARCHITECT®, Abbott Laboratories) may have a relatively shorter
incubation time (e.g., approximately 18 minutes for ARCHITECT®).
Similarly, whereas a non-automated format such as an ELISA may incubate a
detection antibody such as the conjugate reagent for a relatively longer
incubation time (e.g., about 2 hours), an automated or semi-automated
format (e.g., ARCHITECT®) may have a relatively shorter incubation
time (e.g., approximately 4 minutes for the ARCHITECT®).

[0148]Other platforms available from Abbott Laboratories include, but are
not limited to, AxSYM®, IMx® (see, e.g., U.S. Pat. No. 5,294,404,
which is hereby incorporated by reference in its entirety), PRISM®,
EIA (bead), and Quantum® II, as well as other platforms. Additionally,
the assays, kits and kit components can be employed in other formats, for
example, on electrochemical or other hand-held or point-of-care assay
systems. The present disclosure is, for example, applicable to the
commercial Abbott Point of Care (i-STAT®, Abbott Laboratories)
electrochemical immunoassay system that performs sandwich immunoassays.
Immunosensors and their methods of manufacture and operation in
single-use test devices are described, for example in, U.S. Pat. No.
5,063,081, U.S. Pat. App. Pub. No. 2003/0170881, U.S. Pat. App. Pub. No.
2004/0018577, U.S. Pat. App. Pub. No. 2005/0054078, and U.S. Pat. App.
Pub. No. 2006/0160164, which are incorporated in their entireties by
reference for their teachings regarding same.

[0149]In particular, with regard to the adaptation of an autoantibody
assay to the I-STAT® system, the following configuration is
preferred. A microfabricated silicon chip is manufactured with a pair of
gold amperometric working electrodes and a silver-silver chloride
reference electrode. On one of the working electrodes, polystyrene beads
(0.2 mm diameter) with immobilized capture antibody are adhered to a
polymer coating of patterned polyvinyl alcohol over the electrode. This
chip is assembled into an I-STAT® cartridge with a fluidics format
suitable for immunoassay. On a portion of the wall of the sample-holding
chamber of the cartridge there is a layer comprising the second detection
antibody labeled with alkaline phosphatase (or other label). Within the
fluid pouch of the cartridge is an aqueous reagent that includes
p-aminophenol phosphate.

[0150]In operation, a sample suspected of containing NGAL autoantibodies
is added to the holding chamber of the test cartridge and the cartridge
is inserted into the I-STAT® reader. After the second antibody
(detection antibody) has dissolved into the sample, a pump element within
the cartridge forces the sample into a conduit containing the chip. Here
it is oscillated to promote formation of the sandwich between NGAL, NGAL
autoantibody, and the labeled detection antibody. In the penultimate step
of the assay, fluid is forced out of the pouch and into the conduit to
wash the sample off the chip and into a waste chamber. In the final step
of the assay, the alkaline phosphatase label reacts with p-aminophenol
phosphate to cleave the phosphate group and permit the liberated
p-aminophenol to be electrochemically oxidized at the working electrode.
Based on the measured current, the reader is able to calculate the amount
of NGAL autoantibody in the sample by means of an embedded algorithm and
factory-determined calibration curve.

[0151]It further goes without saying that the methods and kits as
described herein necessarily encompass other reagents and methods for
carrying out the immunoassay. For instance, encompassed are various
buffers such as are known in the art and/or which can be readily prepared
or optimized to be employed, e.g., for washing, as a conjugate diluent,
and/or as a calibrator diluent. An exemplary conjugate diluent is
ARCHITECT® conjugate diluent employed in certain kits (Abbott
Laboratories, Abbott Park, Ill.) and containing
2-(N-morpholino)ethanesulfonic acid (MES), a salt, a protein blocker, an
antimicrobial agent, and a detergent. An exemplary calibrator diluent is
ARCHITECT® human calibrator diluent employed in certain kits (Abbott
Laboratories, Abbott Park, Ill.), which comprises a buffer containing
MES, other salt, a protein blocker, and an antimicrobial agent.

EXAMPLES

[0152]The following examples serve to illustrate the present disclosure.
The examples are not intended to limit the scope of the claimed invention
in any way.

[0154]Recombinant NGAL (R&D Systems, Minneapolis, Minn.) was dissolved in
phosphate buffer (0.2 M, pH 8.0) to a concentration of 4 μg/mL.
Microplates (Costar, Corning Life Sciences, Lowell, Mass.) were coated
with the NGAL solution (100 μL/well) for two hours at 38° C.
with mixing at 280 rpm. The plates were drained, and the NGAL solution
was replaced with a solution of heat-inactivated bovine serum albumin
(BSA, 2% w/v in phosphate-buffered saline (PBS), 300 μL/well). The
plates were incubated at 38° C. for 1 hour with mixing at 280 rpm,
and then drained. The plates were then washed (3×) with a solution
of sucrose (2% w/v in PBS, 300 μL/well), drained, and dried under a
stream of dry nitrogen.

[0155]A murine anti-NGAL antibody (R&D Systems, 100 ng/mL) labeled with an
acridinium-9-carboxamide was added to an NGAL-coated microplate (100
μL/well), which was subsequently incubated for 1 hour at 37° C.
and then washed with ARCHITECT® wash buffer (6×, 350 μL).
The microplate was loaded into a Mithras microplate reader (Berthold
Technologies Inc, Oak Ridge, Tenn.) equilibrated at 28° C. The
chemiluminescence signal from each well was recorded for 2 seconds after
the sequential addition of ARCHITECT® pre-trigger solution (100
μL) and ARCHITECT® trigger solution (100 μL). The NGAL-reactive
murine monoclonal antibody conjugate bound specifically to the
NGAL-coated wells, with an average signal of 1,763,268 relative light
units (RLU) and a 1.2% (CV).

Example 2

[0156]This example describes the analysis of test samples of human plasma
for autoantibodies that react with NGAL.

[0157]Frozen normal donor plasma samples were obtained from the Abbott
Laboratories (Abbott Park, Ill.) specimen bank and thawed at 2-8°
C. prior to use. Microplates were prepared as described in Example 1. A
murine anti-human IgG (subtype IgG2b, kappa) was labeled with a
chemiluminescent acridinium-9-carboxamide. This antibody recognized all
human IgG subtypes while having no significant reactivity toward human
IgM or IgA, or rabbit, sheep or goat IgG. The sample (5 μL) was
diluted with AxSYM® Troponin-I ADV preincubation diluent (95 μL)
in the microplate well. After incubating at 37° C. for 2 hours,
the plate was washed with ARCHITECT® wash buffer (6×, 350
μL). The murine anti-human IgG-specific monoclonal antibody-acridinium
conjugate (100 μL) was then added, and the plate was incubated at
37° C. for 1 hour, before a final wash with ARCHITECT® wash
buffer (6×, 350 μL). The microplate was loaded into a Mithras
microplate reader (Berthold Technologies Inc, Oak Ridge, Tenn.)
equilibrated at 28° C. The chemiluminescence signal from each well
was recorded for 2 seconds after the sequential addition of
ARCHITECT® pre-trigger solution (100 μL) and ARCHITECT®
trigger solution (100 μL). General statistics for the population
tested are shown in Table 2 and 3.

Generation of a box-and-whisker plot for 192 samples, in which the
difference between the 25th percentile and the 75th percentile
was 885.0, revealed that the outside value was ≧3,227.5 (17
outside; 8.9%) and the far outside value was ≧4,555.0 (11 outside;
5.7%). These results confirm that, surprisingly and unexpectedly, one or
more autoantibodies reactive with NGAL are found in human samples.

[0158]All patents, patent application publications, journal articles,
textbooks, and other publications mentioned in the specification are
indicative of the level of skill of those in the art to which the
disclosure pertains. All such publications are incorporated herein by
reference to the same extent as if each individual publication were
specifically and individually indicated to be incorporated by reference.

[0159]The invention illustratively described herein may be suitably
practiced in the absence of any element(s) or limitation(s), which is/are
not specifically disclosed herein. Thus, for example, each instance
herein of any of the terms "comprising," "consisting essentially of," and
"consisting of" may be replaced with either of the other two terms.
Likewise, the singular forms "a," "an," and "the" include plural
references unless the context clearly dictates otherwise. Thus, for
example, references to "the method" includes one or more methods and/or
steps of the type, which are described herein and/or which will become
apparent to those ordinarily skilled in the art upon reading the
disclosure.

[0160]The terms and expressions, which have been employed, are used as
terms of description and not of limitation. In this regard, where certain
terms are defined under "Definitions" and are otherwise defined,
described, or discussed elsewhere in the "Detailed Description," all such
definitions, descriptions, and discussions are intended to be attributed
to such terms. There also is no intention in the use of such terms and
expressions of excluding any equivalents of the features shown and
described or portions thereof. Furthermore, while subheadings, e.g.,
"Definitions," are used in the "Detailed Description," such use is solely
for ease of reference and is not intended to limit any disclosure made in
one section to that section only; rather, any disclosure made under one
subheading is intended to constitute a disclosure under each and every
other subheading.

[0161]It is recognized that various modifications are possible within the
scope of the claimed invention. Thus, it should be understood that,
although the present invention has been specifically disclosed in the
context of preferred embodiments and optional features, those skilled in
the art may resort to modifications and variations of the concepts
disclosed herein. Such modifications and variations are considered to be
within the scope of the invention as defined by the appended claims.